Methods of treating liver conditions using notch2 antagonists

ABSTRACT

Methods and compositions for the treatment of liver conditions are provided, such methods and compositions comprising Notch2 antagonists, e.g., anti-Notch2 antibodies. Liver conditions include, but are not limited to, chronic liver disease.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.61/543,483, filed Oct. 5, 2011, the disclosure of which is incorporatedherein by reference as if set forth in its entirety.

FIELD OF THE INVENTION

The present invention relates to methods of treating liver conditionsusing Notch2 antagonists. Compositions for the treatment of suchconditions are also provided.

BACKGROUND

The Notch receptor family is a class of evolutionarily conservedtransmembrane receptors that transmit signals affecting development inorganisms as diverse as sea urchins and humans. Notch receptors andtheir ligands Delta and Serrate (known as Jagged in mammals) aretransmembrane proteins with large extracellular domains that containepidermal growth factor (EGF)-like repeats. The number of Notchparalogues differs between species. For example, there are four Notchreceptors in mammals (Notch1-Notch4), two in Caenorhabditis elegans(LIN-12 and GLP-1) and one in Drosophila melanogaster (Notch). Notchreceptors are proteolytically processed during transport to the cellsurface by a furin-like protease at a site 51, which is N-terminal tothe transmembrane domain, producing an extracellular Notch (ECN) subunitand a Notch transmembrane subunit (NTM). These two subunits remainnon-covalently associated and constitute the mature heterodimericcell-surface receptor.

Notch2 ECN subunits contain 36 N-terminal EGF-like repeats followed bythree tandemly repeated Lin 12/Notch Repeat (LNR) modules that precedethe 51 site. Each LNR module contains three disulfide bonds and a groupof conserved acidic and polar residues predicted to coordinate a calciumion. Within the EGF repeat region lie binding sites for the activatingligands.

The Notch2 NTM comprises an extracellular region (which harbors the S2cleavage site), a transmembrane segment (which harbors the S3 cleavagesite), and a large intracellular portion that includes a RAM23 domain,six ankyrin repeats, a transactivation domain and a carboxy-terminalPEST sequence. Stable association of the ECN and NTM subunits isdependent on a heterodimerization domain (HD) comprising thecarboxy-terminal end of the ECN (termed HD-N) and the extracellularamino-terminal end of NTM (termed HD-C). Before ligand-inducedactivation, Notch is maintained in a resting conformation by a negativeregulatory region (NRR), which comprises the three LNRs and the HDdomain. The crystal structure of the Notch2 NRR is reported in Gordon etal., (2007) Nature Structural & Molecular Biology 14:295-300, 2007.

Binding of a Notch ligand to the ECN subunit initiates two successiveproteolytic cleavages that occur through regulated intramembraneproteolysis. The first cleavage by a metalloprotease (ADAM17) at site S2renders the Notch transmembrane subunit susceptible to a second cleavageat site S3 close to the inner leaflet of the plasma membrane. Site S3cleavage, which is catalyzed by a multiprotein complex containingpresenilin and nicastrin and promoting γ-secretase activity, liberatesthe intracellular portion of the Notch transmembrane subunit, allowingit to translocate to the nucleus and activate transcription of targetgenes. (For review of the proteolytic cleavage of Notch, see, e.g.,Sisodia et al., Nat. Rev. Neurosci. 3:281-290, 2002.)

Five Notch ligands of the Jagged and Delta-like classes have beenidentified in humans (Jagged1 (also termed Serrate1), Jagged2 (alsotermed Serrate2), Delta-like1 (also termed DLL1), Delta-like3 (alsotermed DLL3), and Delta-like4 (also termed DLL4)). Each of the ligandsis a single-pass transmembrane protein with a conserved N-terminalDelta, Serrate, LAG-2 (DSL) motif essential for binding Notch. A seriesof EGF-like modules C-terminal to the DSL motif precede themembrane-spanning segment. Unlike the Notch receptors, the ligands haveshort cytoplasmic tails of 70-215 amino acids at the C-terminus. Inaddition, other types of ligands have been reported (e.g., DNER, NB3,and F3/Contactin). (For review of Notch ligands and ligand-mediatedNotch activation, see, e.g., D′ Souza et al., Oncogene 27:5148-5167,2008.)

The Notch pathway functions during diverse developmental andphysiological processes including those affecting neurogenesis in fliesand vertebrates. In general, Notch signaling is involved in lateralinhibition, lineage decisions, and the establishment of boundariesbetween groups of cells. (See, e.g., Bray, Mol. Cell. Biol. 7:678-679,2006.) A variety of human diseases, including cancers andneurodegenerative disorders have been shown to result from mutations ingenes encoding Notch receptors or their ligands. (See, e.g., Nam et al.,Curr. Opin. Chem. Biol. 6:501-509, 2002.)

Certain anti-Notch2 antagonist antibodies having therapeutic efficacyhave been described. (See U.S. Patent Application Publication No. US2009/0081238 A1, expressly incorporated by reference in its entiretyherein.) For example, such antibodies bind to the negative regulatoryregion (NRR) of Notch2, block Notch2 signaling, and inhibit the growthof melanoma cell lines, diffuse large B-cell lymphoma (DLBCL) celllines, and marginal zone B cells. Certain anti-Notch2 antibodiesdescribed therein bind to the LNR-A domain (the first of the threeLIN12/Notch Repeats) and the HD-C domain of Notch2 NRR.

Adult liver has the capacity to regenerate after injury. It has beenspeculated that biliary-hepatocyte progenitor cells (oval cells) in ornear intrahepatic bile ducts can differentiate into adult hepatocytes(Brues and Marble, J. Exp. Med., 65(1):15 (1937); Zajicek et al., Liver,5(6):293 (1985)), which subsequently mature as they move toward thecentral vein and eventually undergo apoptosis and elimination (Benedettiet al., J. Hepatol., 7(3):319 (1988)). Recent lineage-tracing studieshave supported a role of progenitor cells in liver homeostasis andrepair, but the signals that govern precursor differentiation intohepatocytes are poorly understood. While Notch signaling is known to becritical for the proper formation of the intrahepatic biliary systemduring development (Lozier et al., PLoS One 3(3):e1851 (2008); McCrightet al., Development 129(4):1075 (2002)), it was not known what role, ifany, Notch signaling plays in adult hepatocyte formation and in adulthepatobiliary disease.

Chronic liver disease is marked by gradual destruction of liver tissue,especially of hepatocytes and the functional lobular unit, leading tofibrosis (replacement of liver tissue with scar tissue) and cirrhosis(fibrosis with ineffective nodular regeneration and associated loss ofliver function). Moreover, chronic liver disease often includespathological biliary hyperplasia and may increase the risk of livercancer.

There is a need in the art for further therapeutic methods of treatingliver conditions. The invention described herein meets theabove-described needs and provides other benefits.

SUMMARY

The present invention relates to the treatment of liver conditions usingNotch2 antagonists. The present invention is based, in part, on theobservation that anti-Notch2 NRR antibodies (a) improve liver histologicappearance and hepatocyte function in an acute liver damage model invivo and (b) reduce biliary damage and improve hepatocyte function in achronic liver damage model in vivo.

In one aspect, a method of treating a liver condition characterized byliver damage is provided, the method comprising administering to apatient having such condition an effective amount of a Notch2-specificantagonist. In certain embodiments, the liver condition is chronic liverdisease. In certain embodiments, the liver condition is liver fibrosis.

In any of the above embodiments, the Notch2-specific antagonist may bean anti-Notch2 antagonist antibody. In certain embodiments, theanti-Notch2 antagonist antibody is an anti-Notch2 NRR antibody. In onesuch embodiment, the anti-Notch2 NRR antibody binds to the LNR-A andHD-C domains of Notch2 NRR. In another such embodiment, the anti-Notch2NRR antibody is Antibody D, Antibody D-1, Antibody D-2, or Antibody D-3.In another such embodiment, the anti-Notch2 NRR antibody comprises theheavy and light chain variable region CDRs of Antibody D, Antibody D-1,Antibody D-2, or Antibody D-3. In certain embodiments, the anti-Notch2antagonist antibody is an anti-Notch2 antibody that binds to one or moreEGF-like repeats of Notch2.

The above and further aspects and embodiments of the invention areprovided herein.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A-G show transcriptional profiling of hepatic progenitor cellsand Identification of active Notch signaling in hepatic progenitors invivo.

FIGS. 2A-O show that Notch signaling inhibition promotes hepatocytedifferentiation of hepatic progenitors in vitro.

FIGS. 3A-J show that inhibition of Notch2 signaling in vivo promoteshepatocyte differentiation and improved liver function in chronic andacute liver damage models.

FIGS. 4A-E illustrate a liver progenitor cell isolation strategy.

FIGS. 5A-H show an analysis of oval cell-specific gene expressionsignature.

FIGS. 6A-E show a validation of oval cell gene signature and expressionpattern of putative hepatic stem cell markers.

FIGS. 7A-B show a strategy for in vitro differentiation.

FIGS. 8A-F show the efficacy of anti-Notch2 antibody treatment in vivoand its effect on liver growth and proliferation following partialhepatectomy.

FIGS. 9A-H show the effect of anti-Notch2 antibody treatment on serumhepatobiliary function markers following partial hepatectomy.

FIGS. 10A-F show the effect of anti-Notch2 antibody treatment onhepatobiliary and Notch signaling gene expression following partialhepatectomy.

FIGS. 11A-I show serum hepatobiliary function markers following 4 weeksof antibody administration in normal and DDC-fed mice.

FIG. 12 shows the H1, H2, and H3 heavy chain hypervariable region (HVR)sequences of anti-Notch2 NRR monoclonal antibodies designated AntibodyD, Antibody D-1, Antibody D-2, and Antibody D-3. Amino acid positionsare numbered according to the Kabat numbering system as described below.

FIG. 13 shows the L1, L2, and L3 light chain HVR sequences ofanti-Notch2 NRR monoclonal antibodies designated Antibody D, AntibodyD-1, Antibody D-2, and Antibody D-3. Amino acid positions are numberedaccording to the Kabat numbering system as described below.

FIG. 14 shows an alignment of the heavy chain variable region sequencesof Antibody D, Antibody D-1, Antibody D-2, and Antibody D-3. HVRs areenclosed in boxes.

FIG. 15 shows an alignment of the light chain variable region sequencesof Antibody D, Antibody D-1, Antibody D-2, and Antibody D-3. HVRs areenclosed in boxes.

FIGS. 16A-B show exemplary acceptor human variable heavy (VH) consensusframework sequences for use in practicing the instant invention.Sequence identifiers are as follows:

-   -   human VH subgroup I consensus framework “A” minus Kabat CDRs        (SEQ ID NOs:32, 33, 34, 35).    -   human VH subgroup I consensus frameworks “B,” “C,” and “D” minus        extended hypervariable regions (SEQ ID NOs:36, 37, 34, 35; SEQ        ID NOs:36, 37, 38, 35; and SEQ ID NOs:36, 37, 39, 35).    -   human VH subgroup II consensus framework “A” minus Kabat CDRs        (SEQ ID NOs:40, 41, 42, 35).    -   human VH subgroup II consensus frameworks “B,” “C,” and “D”        minus extended hypervariable regions (SEQ ID NOs:43, 44, 42, 35;        SEQ ID NOs:43, 44, 45, 35; and SEQ ID NOs:43, 44, 46, and 35).    -   human VH subgroup III consensus framework “A” minus Kabat CDRs        (SEQ ID NOs:47, 48, 49, 35).    -   human VH subgroup III consensus frameworks “B,” “C,” and “D”        minus extended hypervariable regions (SEQ ID NOs:50, 51, 49, 35;        SEQ ID NOs:50, 51, 52, 35; and SEQ ID NOs:50, 51, 53, 35).    -   human VH acceptor framework “A” minus Kabat CDRs (SEQ ID NOs:54,        48, 55, 35).    -   human VH acceptor frameworks “B” and “C” minus extended        hypervariable regions (SEQ ID NOs:50, 51, 55, 35; and SEQ ID        NOs:50, 51, 56, 35).    -   human VH acceptor 2 framework “A” minus Kabat CDRs (SEQ ID        NOs:54, 48, 57, 35).    -   human VH acceptor 2 framework “B,” “C,” and “D” minus extended        hypervariable regions (SEQ ID NOs:50, 51, 57, 35; SEQ ID NOs:50,        51, 58, 35; and SEQ ID NOs:50, 51, 59, 35).

FIG. 17 shows exemplary acceptor human variable light (VL) consensusframework sequences for use in practicing the instant invention.Sequence identifiers are as follows:

-   -   human VL kappa subgroup I consensus framework (κv1): SEQ ID        NOs:60, 61, 62, 63    -   human VL kappa subgroup II consensus framework (κv2): SEQ ID        NOs:64, 65, 66, 63    -   human VL kappa subgroup III consensus framework (κv3): SEQ ID        NOs:67, 68, 69, 63    -   human VL kappa subgroup IV consensus framework (κv4): SEQ ID        NOs:70, 71, 72, 63

FIG. 18 shows framework sequences of huMAb4D5-8 light and heavy chains.Numbers in superscript/bold indicate amino acid positions according toKabat.

FIG. 19 shows framework sequences of huMAb4D5-8 light and heavy chainswith the indicated modifications. Numbers in superscript/bold indicateamino acid positions according to Kabat.

DETAILED DESCRIPTION OF EMBODIMENTS I. Definitions

For purposes of interpreting this specification, the followingdefinitions will apply and whenever appropriate, terms used in thesingular will also include the plural and vice versa. In the event thatany definition set forth below conflicts with any document incorporatedherein by reference, the definition set forth below shall control.

The term “Notch,” as used herein, refers, unless specifically orcontextually indicated otherwise, to any native or variant (whethernative or synthetic) Notch polypeptide (Notch1-4). The term “nativesequence” specifically encompasses naturally occurring truncated forms(e.g., an extracellular domain sequence or a transmembrane subunitsequence), naturally occurring variant forms (e.g., alternativelyspliced forms) and naturally-occurring allelic variants. The term“wild-type Notch” generally refers to a polypeptide comprising an aminoacid sequence of a naturally occurring, non-mutated Notch protein. Theterm “wild-type Notch sequence” generally refers to an amino acidsequence found in a naturally occurring, non-mutated Notch.

The term “Notch2,” as used herein, refers, unless specifically orcontextually indicated otherwise, to any native or variant (whethernative or synthetic) Notch2 polypeptide. The term “native sequence”specifically encompasses naturally occurring truncated forms (e.g., anextracellular domain sequence or a transmembrane subunit sequence),naturally occurring variant forms (e.g., alternatively spliced forms)and naturally occurring allelic variants. The term “wild-type Notch2”generally refers to a polypeptide comprising an amino acid sequence of anaturally occurring, non-mutated Notch2 protein. The term “wild typeNotch2 sequence” generally refers to an amino acid sequence found in anaturally occurring, non-mutated Notch2.

The term “Notch2 ligand,” as used herein, refers, unless specifically orcontextually indicated otherwise, to any native or variant (whethernative or synthetic) Notch2 ligand (for example, Jagged1, Jagged2,Delta-like1, Delta-like3, and/or Delta-like4) polypeptide. The term“native sequence” specifically encompasses naturally occurring truncatedforms (e.g., an extracellular domain sequence or a transmembrane subunitsequence), naturally occurring variant forms (e.g., alternativelyspliced forms) and naturally occurring allelic variants. The term“wild-type Notch2 ligand” generally refers to a polypeptide comprisingan amino acid sequence of a naturally occurring, non-mutated Notch2ligand. The term “wild type Notch2 ligand sequence” generally refers toan amino acid sequence found in a naturally occurring, non-mutatedNotch2 ligand.

The term “Notch2 NRR,” as used herein, refers, unless specifically orcontextually indicated otherwise, to any native or variant (whethernative or synthetic) polypeptide region of Notch2 consisting of the 3LNR modules and the amino acid sequences extending from thecarboxy-terminus of the LNR modules to the transmembrane domain, suchsequences including the HD domain (HD-N and HD-C). An exemplary Notch2NRR consists of the region from about amino acid 1422-1677 of humanNotch2 (SEQ ID NO:73). An exemplary human Notch2 NRR is also shown inSEQ ID NO:74. The term “native sequence Notch2 NRR” specificallyencompasses naturally occurring truncated forms, naturally occurringvariant forms (e.g., alternatively spliced forms) andnaturally-occurring allelic variants of a Notch2 NRR. The term“wild-type Notch2 NRR” generally refers to a naturally occurring,non-mutated Notch2 NRR. In some embodiments, a Notch2 NRR is containedin a Notch2, such as, for example, a Notch2 processed at the 51, S2and/or S3 site(s), or an unprocessed Notch2. In some embodiments, aNotch2 NRR contains two or more non-covalently linked fragments of aNotch2 NRR amino acid sequence, e.g., a fragment containing amino acids1422 to 1608 of SEQ ID NO:73 non-covalently linked to a fragmentcontaining amino acids 1609 to 1677 of SEQ ID NO:73.

The term “increased Notch2 signaling,” as used herein, refers to anincrease in Notch2 signaling that is significantly above the level ofNotch2 signaling observed in a control under substantially identicalconditions. In certain embodiments, the increase in Notch2 signaling isat least two fold, three fold, four fold, five fold, or ten fold abovethe level observed in the control. The term “decreased Notch1signaling,” as used herein, refers to a decrease in Notch2 signalingthat is significantly below the level of Notch2 signaling observed in acontrol under substantially identical conditions. In certainembodiments, the decrease in Notch2 signaling is at least two fold,three fold, four fold, five fold, or ten fold below the level observedin the control.

In certain embodiments, Notch2 signaling (i.e., increased or decreasedNotch2 signaling) is assessed using a suitable reporter assay, e.g, asdescribed in U.S. Patent Application Publication No. US 2010/0080808 A1.

The term “anti-Notch2 antibody” or “an antibody that binds to Notch2”refers to an antibody that is capable of binding Notch2 with sufficientaffinity such that the antibody is useful as a diagnostic and/ortherapeutic agent in targeting Notch2. Preferably, the extent of bindingof an anti-Notch2 antibody to an unrelated, non-Notch protein is lessthan about 10% of the binding of the antibody to Notch2 as measured,e.g., by a radioimmunoassay (RIA). In certain embodiments, an antibodythat binds to Notch2 has a dissociation constant (Kd) of ≦1 μM, ≦0.5 μM,≦100 nM, ≦50 nM, ≦10 nM, ≦5 nM, ≦1 nM, ≦0.5 nM, or ≦0.1 nM. In certainembodiments, an anti-Notch2 antibody binds to an epitope of Notch2 thatis conserved among Notch2 from different species, e.g., rodents (mice,rats) and primates.

The term “anti-Notch2 NRR antibody” or “an antibody that binds to Notch2NRR” refers to an antibody that is capable of binding Notch2 NRR withsufficient affinity such that the antibody is useful as a diagnosticand/or therapeutic agent in targeting Notch2. Preferably, the extent ofbinding of an anti-Notch2 NRR antibody to an unrelated, non-Notchprotein is less than about 10% of the binding of the antibody to Notch2NRR as measured, e.g., by a radioimmunoassay (RIA). In certainembodiments, an antibody that binds to Notch2 NRR has a dissociationconstant (Kd) of ≦1 μM, ≦0.5 μM, ≦100 nM, ≦50 nM, ≦10 nM, ≦5 nM, ≦1 nM,≦0.5 nM, or ≦0.1 nM. In certain embodiments, an anti-Notch2 NRR antibodybinds to an epitope of Notch that is conserved among Notch fromdifferent species, e.g., rodents (mice, rats) and primates.

The term “Notch2-specific antagonist” refers to an agent that effectsdecreased Notch2 signaling, as defined above, and does not significantlyaffect signaling by another Notch receptor (Notch1, 3, or 4 in mammals).

An “anti-Notch2 antagonist antibody” is an anti-Notch2 antibody(including an anti-Notch2 NRR antibody) that effects decreased Notch2signaling, as defined above.

The term “antagonist” refers to an agent that significantly inhibits(either partially or completely) the biological activity of a targetmolecule.

The term “antibody” herein is used in the broadest sense andspecifically covers monoclonal antibodies, polyclonal antibodies,multispecific antibodies (e.g. bispecific antibodies) formed from atleast two intact antibodies, and antibody fragments so long as theyexhibit the desired biological activity.

An “isolated” antibody is one which has been identified and separatedand/or recovered from a component of its natural environment.Contaminant components of its natural environment are materials whichwould interfere with research, diagnostic or therapeutic uses for theantibody, and may include enzymes, hormones, and other proteinaceous ornonproteinaceous solutes. In some embodiments, an antibody is purified(1) to greater than 95% by weight of antibody as determined by, forexample, the Lowry method, and in some embodiments, to greater than 99%by weight; (2) to a degree sufficient to obtain at least 15 residues ofN-terminal or internal amino acid sequence by use of, for example, aspinning cup sequenator, or (3) to homogeneity by SDS-PAGE underreducing or nonreducing conditions using, for example, Coomassie blue orsilver stain. Isolated antibody includes the antibody in situ withinrecombinant cells since at least one component of the antibody's naturalenvironment will not be present. Ordinarily, however, isolated antibodywill be prepared by at least one purification step.

“Native antibodies” are usually heterotetrameric glycoproteins of about150,000 daltons, composed of two identical light (L) chains and twoidentical heavy (H) chains. Each light chain is linked to a heavy chainby one covalent disulfide bond, while the number of disulfide linkagesvaries among the heavy chains of different immunoglobulin isotypes. Eachheavy and light chain also has regularly spaced intrachain disulfidebridges. Each heavy chain has at one end a variable domain (V_(H))followed by a number of constant domains. Each light chain has avariable domain at one end (V_(L)) and a constant domain at its otherend; the constant domain of the light chain is aligned with the firstconstant domain of the heavy chain, and the light chain variable domainis aligned with the variable domain of the heavy chain. Particular aminoacid residues are believed to form an interface between the light chainand heavy chain variable domains.

The “variable region” or “variable domain” of an antibody refers to theamino-terminal domains of the heavy or light chain of the antibody. Thevariable domain of the heavy chain may be referred to as “VH.” Thevariable domain of the light chain may be referred to as “VL.” Thesedomains are generally the most variable parts of an antibody and containthe antigen-binding sites.

The term “variable” refers to the fact that certain portions of thevariable domains differ extensively in sequence among antibodies and areused in the binding and specificity of each particular antibody for itsparticular antigen. However, the variability is not evenly distributedthroughout the variable domains of antibodies. It is concentrated inthree segments called hypervariable regions (HVRs) both in thelight-chain and the heavy-chain variable domains. The more highlyconserved portions of variable domains are called the framework regions(FR). The variable domains of native heavy and light chains eachcomprise four FR regions, largely adopting a beta-sheet configuration,connected by three HVRs, which form loops connecting, and in some casesforming part of, the beta-sheet structure. The HVRs in each chain areheld together in close proximity by the FR regions and, with the HVRsfrom the other chain, contribute to the formation of the antigen-bindingsite of antibodies (see Kabat et al., Sequences of Proteins ofImmunological Interest, Fifth Edition, National Institute of Health,Bethesda, Md. (1991)). The constant domains are not involved directly inthe binding of an antibody to an antigen, but exhibit various effectorfunctions, such as participation of the antibody in antibody-dependentcellular toxicity.

The “light chains” of antibodies (immunoglobulins) from any vertebratespecies can be assigned to one of two clearly distinct types, calledkappa (κ) and lambda (λ), based on the amino acid sequences of theirconstant domains.

Depending on the amino acid sequences of the constant domains of theirheavy chains, antibodies (immunoglobulins) can be assigned to differentclasses. There are five major classes of immunoglobulins: IgA, IgD, IgE,IgG, and IgM, and several of these may be further divided intosubclasses (isotypes), e.g., IgG₁, IgG₂, IgG₃, IgG₄, IgA₁, and IgA₂. Theheavy chain constant domains that correspond to the different classes ofimmunoglobulins are called α, δ, ε, γ, and μ, respectively. The subunitstructures and three-dimensional configurations of different classes ofimmunoglobulins are well known and described generally in, for example,Abbas et al. Cellular and Mol. Immunology, 4th ed. (W.B. Saunders, Co.,2000). An antibody may be part of a larger fusion molecule, formed bycovalent or non-covalent association of the antibody with one or moreother proteins or peptides.

The terms “full length antibody,” “intact antibody” and “whole antibody”are used herein interchangeably to refer to an antibody in itssubstantially intact form, not antibody fragments as defined below. Theterms particularly refer to an antibody with heavy chains that containan Fc region.

A “naked antibody” for the purposes herein is an antibody that is notconjugated to a cytotoxic moiety or radiolabel.

“Antibody fragments” comprise a portion of an intact antibody,preferably comprising the antigen binding region thereof. Examples ofantibody fragments include Fab, Fab′, F(ab′)₂, and Fv fragments;diabodies; linear antibodies; single-chain antibody molecules; andmultispecific antibodies formed from antibody fragments.

Papain digestion of antibodies produces two identical antigen-bindingfragments, called “Fab” fragments, each with a single antigen-bindingsite, and a residual “Fc” fragment, whose name reflects its ability tocrystallize readily. Pepsin treatment yields an F(ab′)₂ fragment thathas two antigen-combining sites and is still capable of cross-linkingantigen.

“Fv” is the minimum antibody fragment which contains a completeantigen-binding site. In one embodiment, a two-chain Fv species consistsof a dimer of one heavy- and one light-chain variable domain in tight,non-covalent association. In a single-chain Fv (scFv) species, oneheavy- and one light-chain variable domain can be covalently linked by aflexible peptide linker such that the light and heavy chains canassociate in a “dimeric” structure analogous to that in a two-chain Fvspecies. It is in this configuration that the three HVRs of eachvariable domain interact to define an antigen-binding site on thesurface of the VH-VL dimer. Collectively, the six HVRs conferantigen-binding specificity to the antibody. However, even a singlevariable domain (or half of an Fv comprising only three HVRs specificfor an antigen) has the ability to recognize and bind antigen, althoughat a lower affinity than the entire binding site.

The Fab fragment contains the heavy- and light-chain variable domainsand also contains the constant domain of the light chain and the firstconstant domain (CH1) of the heavy chain. Fab′ fragments differ from Fabfragments by the addition of a few residues at the carboxy terminus ofthe heavy chain CH1 domain including one or more cysteines from theantibody hinge region. Fab′-SH is the designation herein for Fab′ inwhich the cysteine residue(s) of the constant domains bear a free thiolgroup. F(ab′)₂ antibody fragments originally were produced as pairs ofFab′ fragments which have hinge cysteines between them. Other chemicalcouplings of antibody fragments are also known.

“Single-chain Fv” or “scFv” antibody fragments comprise the VH and VLdomains of antibody, wherein these domains are present in a singlepolypeptide chain. Generally, the scFv polypeptide further comprises apolypeptide linker between the VH and VL domains which enables the scFvto form the desired structure for antigen binding. For a review of scFv,see, e.g., Pluckthün, in The Pharmacology of Monoclonal Antibodies, vol.113, Rosenburg and Moore eds., (Springer-Verlag, New York, 1994), pp.269-315.

The term “diabodies” refers to antibody fragments with twoantigen-binding sites, which fragments comprise a heavy-chain variabledomain (VH) connected to a light-chain variable domain (VL) in the samepolypeptide chain (V_(H)-V_(L)). By using a linker that is too short toallow pairing between the two domains on the same chain, the domains areforced to pair with the complementary domains of another chain andcreate two antigen-binding sites. Diabodies may be bivalent orbispecific. Diabodies are described more fully in, for example, EP404,097; WO 1993/01161; Hudson et al., Nat. Med. 9:129-134 (2003); andHollinger et al., Proc. Natl. Acad. Sci. USA 90: 6444-6448 (1993).Triabodies and tetrabodies are also described in Hudson et al., Nat.Med. 9:129-134 (2003).

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies,i.e., the individual antibodies comprising the population are identicalexcept for possible mutations, e.g., naturally occurring mutations, thatmay be present in minor amounts. Thus, the modifier “monoclonal”indicates the character of the antibody as not being a mixture ofdiscrete antibodies. In certain embodiments, such a monoclonal antibodytypically includes an antibody comprising a polypeptide sequence thatbinds a target, wherein the target-binding polypeptide sequence wasobtained by a process that includes the selection of a single targetbinding polypeptide sequence from a plurality of polypeptide sequences.For example, the selection process can be the selection of a uniqueclone from a plurality of clones, such as a pool of hybridoma clones,phage clones, or recombinant DNA clones. It should be understood that aselected target binding sequence can be further altered, for example, toimprove affinity for the target, to humanize the target bindingsequence, to improve its production in cell culture, to reduce itsimmunogenicity in vivo, to create a multispecific antibody, etc., andthat an antibody comprising the altered target binding sequence is alsoa monoclonal antibody of this invention. In contrast to polyclonalantibody preparations, which typically include different antibodiesdirected against different determinants (epitopes), each monoclonalantibody of a monoclonal antibody preparation is directed against asingle determinant on an antigen. In addition to their specificity,monoclonal antibody preparations are advantageous in that they aretypically uncontaminated by other immunoglobulins.

The modifier “monoclonal” indicates the character of the antibody asbeing obtained from a substantially homogeneous population ofantibodies, and is not to be construed as requiring production of theantibody by any particular method. For example, the monoclonalantibodies to be used in accordance with the present invention may bemade by a variety of techniques, including, for example, the hybridomamethod (e.g., Kohler and Milstein, Nature, 256:495-97 (1975); Hongo etal., Hybridoma, 14 (3): 253-260 (1995), Harlow et al., Antibodies: ALaboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988);Hammerling et al., in: Monoclonal Antibodies and T-Cell Hybridomas563-681 (Elsevier, N.Y., 1981)), recombinant DNA methods (see, e.g.,U.S. Pat. No. 4,816,567), phage-display technologies (see, e.g.,Clackson et al., Nature, 352: 624-628 (1991); Marks et al., J. Mol.Biol. 222: 581-597 (1992); Sidhu et al., J. Mol. Biol. 338(2): 299-310(2004); Lee et al., J. Mol. Biol. 340(5): 1073-1093 (2004); Fellouse,Proc. Natl. Acad. Sci. USA 101(34): 12467-12472 (2004); and Lee et al.,J. Immunol. Methods 284(1-2): 119-132 (2004), and technologies forproducing human or human-like antibodies in animals that have parts orall of the human immunoglobulin loci or genes encoding humanimmunoglobulin sequences (see, e.g., WO 1998/24893; WO 1996/34096; WO1996/33735; WO 1991/10741; Jakobovits et al., Proc. Natl. Acad. Sci. USA90: 2551 (1993); Jakobovits et al., Nature 362: 255-258 (1993);Bruggemann et al., Year in Immunol. 7:33 (1993); U.S. Pat. Nos.5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; and 5,661,016;Marks et al., Bio/Technology 10: 779-783 (1992); Lonberg et al., Nature368: 856-859 (1994); Morrison, Nature 368: 812-813 (1994); Fishwild etal., Nature Biotechnol. 14: 845-851 (1996); Neuberger, NatureBiotechnol. 14: 826 (1996); and Lonberg and Huszar, Intern. Rev.Immunol. 13: 65-93 (1995).

The monoclonal antibodies herein specifically include “chimeric”antibodies in which a portion of the heavy and/or light chain isidentical with or homologous to corresponding sequences in antibodiesderived from a particular species or belonging to a particular antibodyclass or subclass, while the remainder of the chain(s) is identical withor homologous to corresponding sequences in antibodies derived fromanother species or belonging to another antibody class or subclass, aswell as fragments of such antibodies, so long as they exhibit thedesired biological activity (see, e.g., U.S. Pat. No. 4,816,567; andMorrison et al., Proc. Natl. Acad. Sci. USA 81:6851-6855 (1984)).Chimeric antibodies include PRIMATIZED® antibodies wherein theantigen-binding region of the antibody is derived from an antibodyproduced by, e.g., immunizing macaque monkeys with the antigen ofinterest.

“Humanized” forms of non-human (e.g., murine) antibodies are chimericantibodies that contain minimal sequence derived from non-humanimmunoglobulin. In one embodiment, a humanized antibody is a humanimmunoglobulin (recipient antibody) in which residues from a HVR of therecipient are replaced by residues from a HVR of a non-human species(donor antibody) such as mouse, rat, rabbit, or nonhuman primate havingthe desired specificity, affinity, and/or capacity. In some instances,FR residues of the human immunoglobulin are replaced by correspondingnon-human residues. Furthermore, humanized antibodies may compriseresidues that are not found in the recipient antibody or in the donorantibody. These modifications may be made to further refine antibodyperformance. In general, a humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the hypervariable loops correspondto those of a non-human immunoglobulin, and all or substantially all ofthe FRs are those of a human immunoglobulin sequence. The humanizedantibody optionally will also comprise at least a portion of animmunoglobulin constant region (Fc), typically that of a humanimmunoglobulin. For further details, see, e.g., Jones et al., Nature321:522-525 (1986); Riechmann et al., Nature 332:323-329 (1988); andPresta, Curr. Op. Struct. Biol. 2:593-596 (1992). See also, e.g.,Vaswani and Hamilton, Ann. Allergy, Asthma & Immunol. 1:105-115 (1998);Harris, Biochem. Soc. Transactions 23:1035-1038 (1995); Hurle and Gross,Curr. Op. Biotech. 5:428-433 (1994); and U.S. Pat. Nos. 6,982,321 and7,087,409.

A “human antibody” is one which possesses an amino acid sequence whichcorresponds to that of an antibody produced by a human and/or has beenmade using any of the techniques for making human antibodies asdisclosed herein. This definition of a human antibody specificallyexcludes a humanized antibody comprising non-human antigen-bindingresidues. Human antibodies can be produced using various techniquesknown in the art, including phage-display libraries. Hoogenboom andWinter, J. Mol. Biol., 227:381 (1991); Marks et al., J. Mol. Biol.,222:581 (1991). Also available for the preparation of human monoclonalantibodies are methods described in Cole et al., Monoclonal Antibodiesand Cancer Therapy, Alan R. Liss, p. 77 (1985); Boerner et al., J.Immunol., 147(1):86-95 (1991). See also van Dijk and van de Winkel,Curr. Opin. Pharmacol., 5: 368-74 (2001). Human antibodies can beprepared by administering the antigen to a transgenic animal that hasbeen modified to produce such antibodies in response to antigenicchallenge, but whose endogenous loci have been disabled, e.g., immunizedxenomice (see, e.g., U.S. Pat. Nos. 6,075,181 and 6,150,584 regardingXENOMOUSE™ technology). See also, for example, Li et al., Proc. Natl.Acad. Sci. USA, 103:3557-3562 (2006) regarding human antibodiesgenerated via a human B-cell hybridoma technology.

The term “hypervariable region,” “HVR,” or “HV,” when used herein refersto the regions of an antibody variable domain which are hypervariable insequence and/or form structurally defined loops. Generally, antibodiescomprise six HVRs; three in the VH (H1, H2, H3), and three in the VL(L1, L2, L3). In native antibodies, H3 and L3 display the most diversityof the six HVRs, and H3 in particular is believed to play a unique rolein conferring fine specificity to antibodies. See, e.g., Xu et al.,Immunity 13:37-45 (2000); Johnson and Wu, in Methods in MolecularBiology 248:1-25 (Lo, ed., Human Press, Totowa, N.J., 2003). Indeed,naturally occurring camelid antibodies consisting of a heavy chain onlyare functional and stable in the absence of light chain. See, e.g.,Hamers-Casterman et al., Nature 363:446-448 (1993); Sheriff et al.,Nature Struct. Biol. 3:733-736 (1996).

A number of HVR delineations are in use and are encompassed herein. TheKabat Complementarity Determining Regions (CDRs) are based on sequencevariability and are the most commonly used (Kabat et al., Sequences ofProteins of Immunological Interest, 5th Ed. Public Health Service,National Institutes of Health, Bethesda, Md. (1991)). Chothia refersinstead to the location of the structural loops (Chothia and Lesk J.Mol. Biol. 196:901-917 (1987)). The AbM HVRs represent a compromisebetween the Kabat HVRs and Chothia structural loops, and are used byOxford Molecular's AbM antibody modeling software. The “contact” HVRsare based on an analysis of the available complex crystal structures.The residues from each of these HVRs are noted below.

Loop Kabat AbM Chothia Contact L1 L24-L34 L24-L34 L26-L32 L30-L36 L2L50-L56 L50-L56 L50-L52 L46-L55 L3 L89-L97 L89-L97 L91-L96 L89-L96 H1H31-H35B H26-H35B H26-H32 H30-H35B (Kabat Numbering) H1 H31-H35 H26-H35H26-H32 H30-H35 (Chothia Numbering) H2 H50-H65 H50-H58 H53-H55 H47-H58H3 H95-H102 H95-H102 H96-H101 H93-H101

HVRs may comprise “extended HVRs” as follows: 24-36 or 24-34 (L1), 46-56or 50-56 (L2) and 89-97 or 89-96 (L3) in the VL and 26-35 (H1), 50-65 or49-65 (H2) and 93-102, 94-102, or 95-102 (H3) in the VH. The variabledomain residues are numbered according to Kabat et al., supra, for eachof these definitions.

“Framework” or “FR” residues are those variable domain residues otherthan the HVR residues as herein defined.

The term “variable domain residue numbering as in Kabat” or “amino acidposition numbering as in Kabat,” and variations thereof, refers to thenumbering system used for heavy chain variable domains or light chainvariable domains of the compilation of antibodies in Kabat et al.,supra. Using this numbering system, the actual linear amino acidsequence may contain fewer or additional amino acids corresponding to ashortening of, or insertion into, a FR or HVR of the variable domain.For example, a heavy chain variable domain may include a single aminoacid insert (residue 52a according to Kabat) after residue 52 of H2 andinserted residues (e.g. residues 82a, 82b, and 82c, etc. according toKabat) after heavy chain FR residue 82. The Kabat numbering of residuesmay be determined for a given antibody by alignment at regions ofhomology of the sequence of the antibody with a “standard” Kabatnumbered sequence.

The Kabat numbering system is generally used when referring to a residuein the variable domain (approximately residues 1-107 of the light chainand residues 1-113 of the heavy chain) (e.g., Kabat et al., supra). The“EU numbering system” or “EU index” is generally used when referring toa residue in an immunoglobulin heavy chain constant region (e.g., the EUindex reported in Kabat et al., supra). The “EU index as in Kabat”refers to the residue numbering of the human IgG1 EU antibody. Unlessstated otherwise herein, references to residue numbers in the variabledomain of antibodies means residue numbering by the Kabat numberingsystem. Unless stated otherwise herein, references to residue numbers inthe constant domain of antibodies means residue numbering by the EUnumbering system (e.g., see United States Patent Application PublicationUS 2008/0181888 A1, Figures for EU numbering).

An “affinity matured” antibody is one with one or more alterations inone or more HVRs thereof which result in an improvement in the affinityof the antibody for antigen, compared to a parent antibody which doesnot possess those alteration(s). In one embodiment, an affinity maturedantibody has nanomolar or even picomolar affinities for the targetantigen. Affinity matured antibodies may be produced using certainprocedures known in the art. For example, Marks et al. Bio/Technology10:779-783 (1992) describe affinity maturation by VH and VL domainshuffling. Random mutagenesis of HVR and/or framework residues isdescribed by, for example, in Barbas et al. Proc Nat. Acad. Sci. USA91:3809-3813 (1994); Schier et al. Gene 169:147-155 (1995); Yelton etal. J. Immunol. 155:1994-2004 (1995); Jackson et al., J. Immunol.154(7):3310-9 (1995); and Hawkins et al, J. Mol. Biol. 226:889-896(1992).

Antibody “effector functions” refer to those biological activitiesattributable to the Fc region (a native sequence Fc region or amino acidsequence variant Fc region) of an antibody, and vary with the antibodyisotype. Examples of antibody effector functions include: C1q bindingand complement dependent cytotoxicity (CDC); Fc receptor binding;antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; downregulation of cell surface receptors (e.g. B cell receptor); and B cellactivation.

“Binding affinity” generally refers to the strength of the sum total ofnoncovalent interactions between a single binding site of a molecule(e.g., an antibody) and its binding partner (e.g., an antigen). Unlessindicated otherwise, as used herein, “binding affinity” refers tointrinsic binding affinity which reflects a 1:1 interaction betweenmembers of a binding pair (e.g., antibody and antigen). The affinity ofa molecule X for its partner Y can generally be represented by thedissociation constant (Kd). Affinity can be measured by common methodsknown in the art, including those described herein. Low-affinityantibodies generally bind antigen slowly and tend to dissociate readily,whereas high-affinity antibodies generally bind antigen faster and tendto remain bound longer. A variety of methods of measuring bindingaffinity are known in the art, any of which can be used for purposes ofthe present invention. Specific illustrative and exemplary embodimentsfor measuring binding affinity are described in the following.

In one embodiment, the “Kd” or “Kd value” according to this invention ismeasured by a radiolabeled antigen binding assay (RIA) performed withthe Fab version of an antibody of interest and its antigen as describedby the following assay. Solution binding affinity of Fabs for antigen ismeasured by equilibrating Fab with a minimal concentration of(¹²⁵I)-labeled antigen in the presence of a titration series ofunlabeled antigen, then capturing bound antigen with an anti-Fabantibody-coated plate (see, e.g., Chen, et al., J. Mol. Biol.293:865-881 (1999)). To establish conditions for the assay, MICROTITER®multi-well plates (Thermo Scientific) are coated overnight with 5 μg/mlof a capturing anti-Fab antibody (Cappel Labs) in 50 mM sodium carbonate(pH 9.6), and subsequently blocked with 2% (w/v) bovine serum albumin inPBS for two to five hours at room temperature (approximately 23° C.). Ina non-adsorbent plate (Nunc #269620), 100 pM or 26 pM [¹²⁵I]-antigen aremixed with serial dilutions of a Fab of interest (e.g., consistent withassessment of the anti-VEGF antibody, Fab-12, in Presta et al., CancerRes. 57:4593-4599 (1997)). The Fab of interest is then incubatedovernight; however, the incubation may continue for a longer period(e.g., about 65 hours) to ensure that equilibrium is reached.Thereafter, the mixtures are transferred to the capture plate forincubation at room temperature (e.g., for one hour). The solution isthen removed and the plate washed eight times with 0.1% TWEEN-20™ inPBS. When the plates have dried, 150 μl/well of scintillant(MICROSCINT-20™; Packard) is added, and the plates are counted on aTOPCOUNT™ gamma counter (Packard) for ten minutes. Concentrations ofeach Fab that give less than or equal to 20% of maximal binding arechosen for use in competitive binding assays. According to anotherembodiment, the Kd or Kd value is measured by using surface plasmonresonance assays using a BIACORE®-2000 or a BIACORE®-3000 (BIAcore,Inc., Piscataway, N.J.) at 25° C. with immobilized antigen CM5 chips at˜10 response units (RU). Briefly, carboxymethylated dextran biosensorchips (CM5, BIACORE, Inc.) are activated withN-ethyl-N′-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) andN-hydroxysuccinimide (NHS) according to the supplier's instructions.Antigen is diluted with 10 mM sodium acetate, pH 4.8, to 5 μg/ml (˜0.2μM) before injection at a flow rate of 5 μl/minute to achieveapproximately 10 response units (RU) of coupled protein. Following theinjection of antigen, 1 M ethanolamine is injected to block unreactedgroups. For kinetics measurements, two-fold serial dilutions of Fab(0.78 nM to 500 nM) are injected in PBS with 0.05% TWEEN-20™ surfactant(PBST) at 25° C. at a flow rate of approximately 25 μl/min. Associationrates (k_(on)) and dissociation rates (k_(off)) are calculated using asimple one-to-one Langmuir binding model (BIACORE® Evaluation Softwareversion 3.2) by simultaneously fitting the association and dissociationsensorgrams. The equilibrium dissociation constant (Kd) is calculated asthe ratio k_(off)/k_(on). See, e.g., Chen et al., J. Mol. Biol.293:865-881 (1999). If the on-rate exceeds 10⁶ M⁻¹ s⁻¹ by the surfaceplasmon resonance assay above, then the on-rate can be determined byusing a fluorescent quenching technique that measures the increase ordecrease in fluorescence emission intensity (excitation=295 nm;emission=340 nm, 16 nm band-pass) at 25° C. of a 20 nM anti-antigenantibody (Fab form) in PBS, pH 7.2, in the presence of increasingconcentrations of antigen as measured in a spectrometer, such as astop-flow equipped spectrophometer (Aviv Instruments) or a 8000-seriesSLM-AMINCO™ spectrophotometer (ThermoSpectronic) with a stirred cuvette.

An “on-rate,” “rate of association,” “association rate,” or “k_(on)”according to this invention can also be determined as described aboveusing a BIACORE®-2000 or a BIACORE®-3000 system (BIAcore, Inc.,Piscataway, N.J.).

The term “substantially similar” or “substantially the same,” as usedherein, denotes a sufficiently high degree of similarity between twonumeric values (for example, one associated with an antibody of theinvention and the other associated with a reference/comparatorantibody), such that one of skill in the art would consider thedifference between the two values to be of little or no biologicaland/or statistical significance within the context of the biologicalcharacteristic measured by said values (e.g., Kd values). The differencebetween said two values is, for example, less than about 50%, less thanabout 40%, less than about 30%, less than about 20%, and/or less thanabout 10% as a function of the reference/comparator value.

The phrase “substantially reduced,” or “substantially different,” asused herein, denotes a sufficiently high degree of difference betweentwo numeric values (generally one associated with a molecule and theother associated with a reference/comparator molecule) such that one ofskill in the art would consider the difference between the two values tobe of statistical significance within the context of the biologicalcharacteristic measured by said values (e.g., Kd values). The differencebetween said two values is, for example, greater than about 10%, greaterthan about 20%, greater than about 30%, greater than about 40%, and/orgreater than about 50% as a function of the value for thereference/comparator molecule.

An “acceptor human framework” or a “human acceptor framework” for thepurposes herein is a framework comprising the amino acid sequence of aVL or VH framework derived from a human immunoglobulin framework or ahuman consensus framework. An acceptor human framework “derived from” ahuman immunoglobulin framework or a human consensus framework maycomprise the same amino acid sequence thereof, or it may containpre-existing amino acid sequence changes. In some embodiments, thenumber of pre-existing amino acid changes are 10 or less, 9 or less, 8or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, or 2 orless. Where pre-existing amino acid changes are present in a VH,preferably those changes occur at only three, two, or one of positions71H, 73H and 78H; for instance, the amino acid residues at thosepositions may be 71A, 73T and/or 78A. In one embodiment, the VL acceptorhuman framework is identical in sequence to the VL human immunoglobulinframework sequence or human consensus framework sequence.

A “human consensus framework” is a framework which represents the mostcommonly occurring amino acid residues in a selection of humanimmunoglobulin VL or VH framework sequences. Generally, the selection ofhuman immunoglobulin VL or VH sequences is from a subgroup of variabledomain sequences. Generally, the subgroup of sequences is a subgroup asin Kabat et al., supra. In one embodiment, for the VL, the subgroup issubgroup kappa I as in Kabat et al., supra. In one embodiment, for theVH, the subgroup is subgroup III as in Kabat et al., supra.

A “VH subgroup III consensus framework” comprises the consensus sequenceobtained from the amino acid sequences in variable heavy subgroup III ofKabat et al., supra. In one embodiment, a human acceptor framework isderived from the VH subgroup III consensus framework and comprises anamino acid sequence comprising at least a portion or all of each of thefollowing sequences: (SEQ ID NO:50)-H1-(SEQ ID NO:51)-H2-(SEQ ID NO:57or 59)-H3-(SEQ ID NO: 35). In some embodiments, the last residue (S11)of SEQ ID NO:35 is substituted with an alanine

A “VL subgroup I consensus framework” comprises the consensus sequenceobtained from the amino acid sequences in variable light kappa subgroupI of Kabat et al., supra. In one embodiment, the VH subgroup I consensusframework amino acid sequence comprises at least a portion or all ofeach of the following sequences: (SEQ ID NO:60)-L1-(SEQ IDNO:61)-L2-(SEQ ID NO:62)-L3-(SEQ ID NO:63).

A “disorder” is any condition or disease that would benefit fromtreatment with a composition or method of the invention. This includeschronic and acute disorders including those pathological conditionswhich predispose the mammal to the disorder in question. Non-limitingexamples of disorders to be treated herein include conditions such ascancer.

The terms “cell proliferative disorder” and “proliferative disorder”refer to disorders that are associated with some degree of abnormal cellproliferation. In one embodiment, the cell proliferative disorder iscancer.

“Tumor,” as used herein, refers to all neoplastic cell growth andproliferation, whether malignant or benign, and all pre-cancerous andcancerous cells and tissues. The terms “cancer,” “cancerous,” “cellproliferative disorder,” “proliferative disorder,” and “tumor” are notmutually exclusive as referred to herein.

A cancer that “responds” to a therapeutic agent is one that shows asignificant decrease in cancer or tumor progression, including but notlimited to, (1) inhibition, to some extent, of tumor growth, includingslowing down and complete growth arrest; (2) reduction in the number ofcancer or tumor cells; (3) reduction in tumor size; (4) inhibition(i.e., reduction, slowing down or complete stopping) of cancer cellinfiltration into adjacent peripheral organs and/or tissues; and/or (5)inhibition (i.e. reduction, slowing down or complete stopping) ofmetastasis.

As used herein, “treatment” (and variations such as “treat” or“treating”) refers to clinical intervention in an attempt to alter thenatural course of the individual or cell being treated, and can beperformed either for prophylaxis or during the course of clinicalpathology. Desirable effects of treatment include preventing occurrenceor recurrence of disease, alleviation of symptoms, diminishment of anydirect or indirect pathological consequences of the disease, preventingmetastasis, decreasing the rate of disease progression, amelioration orpalliation of the disease state, and remission or improved prognosis. Insome embodiments, Notch2 antagonists of the invention are used to delaydevelopment of a disease or disorder or to slow the progression of adisease or disorder.

An “individual,” “subject,” or “patient” is a vertebrate. In certainembodiments, the vertebrate is a mammal. Mammals include, but are notlimited to, farm animals (such as cows), sport animals, pets (such ascats, dogs, and horses), primates, mice and rats. In certainembodiments, a mammal is a human.

The term “pharmaceutical formulation” refers to a preparation which isin such form as to permit the biological activity of the activeingredient to be effective, and which contains no additional componentswhich are unacceptably toxic to a subject to which the formulation wouldbe administered. Such formulations may be sterile.

An “effective amount” refers to an amount effective, at dosages and forperiods of time necessary, to achieve the desired therapeutic orprophylactic result.

The term “progenitor,” “hepatic progenitor,” “liver progenitor” or “ovalcell” refers to small epithelial cells that can differentiate into bothhepatocytes and intra-hepatic bile duct cells.

II. Embodiments of the Invention

The present invention relates to the treatment of liver conditions usingNotch2 antagonists. The present invention is based, in part, on theobservation that anti-Notch2 NRR antibodies (a) improve liver appearanceand hepatocyte function in an acute liver damage model in vivo and (b)reduce biliary damage and improve hepatocyte function in a chronic liverdamage model in vivo. Without being bound by any particular theory oroperation, the Notch2 antagonist might improve liver conditions bypromoting hepatocyte differentiation and/or by decreasing aberrant bileduct proliferation.

In various aspects of the invention, a method of treating a livercondition characterized by liver damage is provided, the methodcomprising administering to a patient having such condition an effectiveamount of a Notch2-specific antagonist. In certain embodiments, theliver condition is chronic liver disease, including but not limited tofibrosis, cirrhosis, viral hepatitis (e.g., hepatitis A, B, C, D, E, orG), autoimmune liver diseases (e.g., autoimmune hepatitis, primarybiliary cirrhosis, or primary sclerosing cholangitis), genetic liverdiseases (e.g., alpha-1 antitrypsin deficiency, Crigler-Najjar syndrome,familial amyloidosis, Gilbert's syndrome, Dubin-Johnson syndrome,hereditary hemchromatosis, primary oxalosis, or Wilson's disease),alcoholic hepatitis or nonalcoholic fatty liver disease. In certainembodiments, the liver condition is an acute liver condition, such asacute liver failure, acute liver injury, or acute liver toxicity, e.g.,acetaminophen toxicity. In certain embodiments, the liver condition isliver cancer, e.g., hepatocellular carcinoma (HCC), intrahepaticcholangiocarcinoma (bile duct cancer), or hepatoblastoma.

In some embodiments, treatment results in improved liver histologicalappearance, including but not limited to, e.g., larger cell size, lowernuclear-to-cytoplasmic ratio, two nuclei, as compared to cell size,nuclear-to-cytoplasmic ration and nuclei number in cultured adult ovalcells. In some embodiments, treatment results in a more differentiatedmorphology, e.g., as compared to morphology of cultured adult ovalcells.

In some embodiments, treatment results in decreased expression ofKeratin-19 biomarker in liver cells, e.g., decreased expression relativeto expression of Keratin-19 biomarker in cultured adult oval cells.Methods for detecting keratin-19 biomarker (e.g., Keratin-19 geneexpression, e.g., mRNA expression) are well known in the art and arealso exemplified herein.

In some embodiments, treatment results in increased expression ofalbumin and AFP e.g., increased expression relative to expression ofalbumin and/or AFP biomarkers in cultured adult oval cells. Methods fordetecting albumin and/or AFP biomarkers (e.g., gene expression, e.g.,mRNA expression) are well known in the art and are also exemplifiedherein.

In some embodiments, treatment results in a reduced number of Hes1positive intrahepatic bile duct cells.

In some embodiment, treatment results in reduced liver progenitor cells(e.g., adult liver oval cell) proliferation within the bile ducts.Reduced proliferation may be determined, e.g., by determining averagecross-sectional area of K19-positive tissue as compared to the totalliver cross sectional area.

In some embodiments, treatment results in improved hepatocyte function.Hepatocyte function may be measured by methods known in the art,including but not limited to: no significant elevation of biomarkersassociated with biliary dysfunction, such as those biomarkers describedin FIG. 11. In some embodiments, a biomarker associated with biliarydysfunction is total and/or direct serum bilirubin level. In someembodiments, a biomarker associated with biliary dysfunction is thedifferentiation quotient, as further described and exemplified herein.

In some embodiments, improved hepatocyte function is determined, e.g.,by assessment of heptobiliary function biomarkers, including but notlimited to the serum heptobiliary function biomarker described in FIGS.2 and 5. In some embodiments, serum heptobiliary function biomarker isserum albumin level.

In some embodiments, improved hepatocyte function is increased rate ofrecovery of liver function.

The invention also provides methods for promoting hepatocytedifferentiation and/or by decreasing aberrant bile duct proliferation,the method comprising administering to a patient in need of suchtreatment an effective amount of a Notch2-specific antagonist. In someembodiments, the patient has a liver condition characterized by liverdamage. In certain embodiments, the liver condition is chronic liverdisease, including but not limited to fibrosis, cirrhosis, viralhepatitis (e.g., hepatitis A, B, C, D, E, or G), autoimmune liverdiseases (e.g., autoimmune hepatitis, primary biliary cirrhosis, orprimary sclerosing cholangitis), genetic liver diseases (e.g., alpha-1antitrypsin deficiency, Crigler-Najjar syndrome, familial amyloidosis,Gilbert's syndrome, Dubin-Johnson syndrome, hereditary hemchromatosis,primary oxalosis, or Wilson's disease), alcoholic hepatitis ornonalcoholic fatty liver disease. In certain embodiments, the livercondition is an acute liver condition, such as acute liver failure,acute liver injury, or acute liver toxicity, e.g., acetaminophentoxicity. In certain embodiments, the liver condition is liver cancer,e.g., hepatocellular carcinoma (HCC), intrahepatic cholangiocarcinoma(bile duct cancer), or hepatoblastoma. In some embodiment, treatmentresults in reduced liver progenitor cell (e.g., adult liver oval cell)proliferation. Reduced proliferation may be determined, e.g., bydetermining average cross-sectional area of K19 positive tissue ascompared to the total liver cross sectional area.

The invention also provides methods for improving liver histologicalappearance, the method comprising administering to a patient in need ofsuch treatment an effective amount of a Notch2-specific antagonist. Insome embodiments, treatment results in improved liver histologicalappearance, including but not limited to: larger cell size, lowernuclear-to-cytoplasmic ratio, two nucleic, e.g., as compared to cellsize, nuclear-to-cytoplasmic ratio and nuclei number in cultured adultoval cells. In some embodiments, treatment results in a moredifferentiated morphology, e.g., as compared to morphology of culturedadult oval cells.

In some embodiments, treatment results in decreased expression ofKeratin-19 biomarker in liver cells, e.g., decreased expression relativeto expression of Keratin-19 biomarker in cultured adult oval cells.Methods for detecting keratin-19 biomarker (e.g., Keratin-19 geneexpression, e.g., mRNA expression) are well known in the art and arealso exemplified herein.

In some embodiments, treatment results in increased expression ofalbumin and AFP e.g., increased expression relative to expression ofalbumin and/or AFP biomarkers in cultured adult oval cells. Methods fordetecting albumin and/or AFP biomarkers (e.g., gene expression, e.g.,mRNA expression) are well known in the art and are also exemplifiedherein.

In some embodiments, treatment results in reduced number of Hes1positive intrahepatic bile duct cells.

The invention also provides methods for reducing serum bile acids, serumbilirubin, serum alkaline phosphatase, serum ALT, and/or serum ASTfollowing hepatic injury, the method comprising administering to apatient in need thereof an effective amount of a Notch2-specificantagonist.

The invention also provides methods for reducing the number ofCK19-positive cells in cell population that comprises an oval cell, themethod comprising the step of contacting the oval cell with aNotch2-specific antagonist.

The invention also provides methods for reducing the expression orsecretion of bile acids, bilirubin, alkaline phosphatase, ALT, and/orAST, the method comprising contacting an oval cell with an effectiveamount of a Notch2-specific antagonist.

The invention provides methods for identifying a patient eligible forreceiving treatment of a liver condition characterized by liver damageby administering to a patient having such condition an effective amountof a Notch2-specific antagonist, the method comprising determiningexpression of one or more of the genes listed in Table 2 in a sampleobtained from the patient. In some embodiments, the genes belong to theNotch pathway, e.g., JAG1. In some embodiments, a sample or biopsy fromthe patient is analyzed for mRNA expression of one of the genes listedin Table 1 using methods well known in the art, such as, e.g.,quantitative PCR analysis, and compared to expression of the same geneor genes in a biopsy obtained from a control individual or compared to areference value. In some embodiments, elevated expression of one or moregenes listed in Table 1 in the biopsy obtained from the patient,relative to the control, identifies the patient as suitable forreceiving treatment with a Notch2-specific antagonist, as describedherein. In some embodiments, additional parameters, such as, e.g.,examination by a physician, histologic evaluation of a biopsy,determination of serum levels indicative of liver damage, etc. areemployed to identify the patient for receiving the treatment. Also,elevated hepatic expression by a patient of one or more of the genesidentified in Table 2 is specifically contemplated as one possibleembodiment of any of the methods provided herein.

In some embodiments patients are selected for treatment with aNotch2-specific antagonist as described herein by measuring other knownmarkers of oval cells or aggressive HCC (see, e.g., Woo et al 2011 Mol.Carcinog. 2011 April; 50(4):235-43). In some embodiments, patients areselected for treatment by analyzing hepatic Notch2 activation, forexample by detection of the activated form of Notch2 as describedherein.

Examples of Notch2-specific antagonists include, but are not limited to,soluble Notch receptors, soluble Notch ligand variants, e.g., dominantnegative ligand variants, aptamers or oligopeptides that bind Notch2 orNotch2 ligands, organic or inorganic molecules that interferespecifically with Notch2 signaling, anti-Notch2 antagonist antibodiesand anti-Notch2 ligand antagonist antibodies. Examples ofNotch2-specific antagonists include those described in U.S. PatentApplication Publication No. US 2010/0111958.

In certain embodiments, the Notch2-specific antagonist is an anti-Notch2antagonist antibody. In one such embodiment, the anti-Notch2 antagonistantibody is an antibody that binds to the extracellular domain of Notch2and effects decreased Notch2 signaling. In one such embodiment, theanti-Notch2 antagonist antibody is an anti-Notch2 NRR antibody.Anti-Notch2 NRR antibodies include, but are not limited to, any of theanti-Notch2 NRR antibodies disclosed in U.S. Patent ApplicationPublication No. US 2010/0080808 A1, which is expressly incorporated byreference herein in its entirety. Such antibodies include, but are notlimited to anti-Notch2 NRR antibodies that bind to the LNR-A and HD-Cdomains of Notch2 NRR. Exemplary anti-Notch2 NRR antibodies aremonoclonal antibodies designated Antibody D, Antibody D-1, Antibody D-2,or Antibody D-3 that were derived from a phage library, as disclosed inUS 2010/0080808. Antibody D that binds to Notch2 NRR was isolated. Thatantibody was affinity matured to generate Antibody D-1, Antibody D-2,and Antibody D-3. The sequences of the heavy chain and light chainhypervariable regions (HVRs) of Antibody D, Antibody D-1, Antibody D-2,and Antibody D-3 are shown in FIGS. 12 and 13. The sequences of theheavy and light chain variable domains of Antibody D, Antibody D-1,Antibody D-2, and Antibody D-3 are shown in FIGS. 14 and 15. Furtherembodiments of anti-Notch2 NRR antibodies are provided as follows.

In one aspect, an antagonist antibody that specifically binds to Notch2NRR is provided, wherein the antibody comprises at least one, two,three, four, five, or six HVRs selected from:

-   -   (a) an HVR-H1 comprising an amino acid sequence that conforms to        the consensus sequence of SEQ ID NO:3;    -   (b) an HVR-H2 comprising the amino acid sequence of SEQ ID NO:4;    -   (c) an HVR-H3 comprising the amino acid sequence of SEQ ID NO:5;    -   (d) an HVR-L1 comprising an amino acid sequence that conforms to        the consensus sequence of SEQ ID NO:10;    -   (e) an HVR-L2 comprising an amino acid sequence that conforms to        the consensus sequence of SEQ ID NO:14; and    -   (f) an HVR-L3 comprising an amino acid sequence that conforms to        the consensus sequence of SEQ ID NO:19.

In a further aspect, the antibody comprises an HVR-H3 comprising theamino acid sequence of SEQ ID NO:5 and at least one, two, three, four,or five HVRs selected from (a), (b), (d), (e), and (f) above. In afurther aspect, the antibody comprises (a), (b), (c), (d), (e), and (f)above. With respect to (a), (d), (e), and (f), any one or more of thefollowing embodiments are contemplated: HVR-H1 comprises an amino acidsequence selected from SEQ ID NOs:1-2; HVR-L1 comprises an amino acidsequence selected from SEQ ID NOs:6-9; HVR-L2 comprises an amino acidsequence selected from SEQ ID NOs:11-13; and HVR-L3 comprises an aminoacid sequence selected from SEQ ID NOs:15-18.

In another aspect, an antibody that specifically binds to Notch2 NRR isprovided, wherein the antibody comprises an HVR-H1 comprising an aminoacid sequence that conforms to the consensus sequence of SEQ ID NO:3, anHVR-H2 comprising the amino acid sequence of SEQ ID NO:4, and an HVR-H3comprising the amino acid sequence of SEQ ID NO:5. In one embodiment,HVR-H1 comprises an amino acid sequence selected from SEQ ID NOs:1-2.

In another aspect, an antibody that specifically binds to Notch2 NRR isprovided, wherein the antibody comprises an HVR-L1 comprising an aminoacid sequence that conforms to the consensus sequence of SEQ ID NO:10,an HVR-L2 comprising an amino acid sequence that conforms to theconsensus sequence of SEQ ID NO:14, and an HVR-L3 comprising an aminoacid sequence that conforms to the consensus sequence of SEQ ID NO:19.The following embodiments are contemplated in any combination: HVR-L1comprises an amino acid sequence selected from SEQ ID NOs:6-9; HVR-L2comprises an amino acid sequence selected from SEQ ID NOs:11-13; andHVR-L3 comprises an amino acid sequence selected from SEQ ID NOs:15-18.In one embodiment, an antibody that binds to Notch2 NRR comprises anHVR-L1 comprising the amino acid sequence of SEQ ID NO:6; an HVR-L2comprising the amino acid sequence of SEQ ID NO:11; and an HVR-L3comprising the amino acid sequence of SEQ ID NO:15. In anotherembodiment, an antibody that binds to Notch2 NRR comprises an HVR-L1comprising the amino acid sequence of SEQ ID NO:7; an HVR-L2 comprisingthe amino acid sequence of SEQ ID NO:11; and an HVR-L3 comprising theamino acid sequence of SEQ ID NO:16. In another embodiment, an antibodythat binds to Notch2 NRR comprises an HVR-L1 comprising the amino acidsequence of SEQ ID NO:8; an HVR-L2 comprising the amino acid sequence ofSEQ ID NO:12; and an HVR-L3 comprising the amino acid sequence of SEQ IDNO:17. In another embodiment, an antibody that binds to Notch2 NRRcomprises an HVR-L1 comprising the amino acid sequence of SEQ ID NO:9;an HVR-L2 comprising the amino acid sequence of SEQ ID NO:13; and anHVR-L3 comprising the amino acid sequence of SEQ ID NO:18.

In one embodiment, an antibody that specifically binds to Notch2 NRR isprovided, wherein the antibody comprises:

-   -   (a) an HVR-H1 comprising the amino acid sequence of SEQ ID NO:1;    -   (b) an HVR-H2 comprising the amino acid sequence of SEQ ID NO:4;    -   (c) an HVR-H3 comprising the amino acid sequence of SEQ ID NO:5;    -   (d) an HVR-L1 comprising the amino acid sequence of SEQ ID NO:6;    -   (e) an HVR-L2 comprising the amino acid sequence of SEQ ID        NO:11; and    -   (f) an HVR-L3 comprising the amino acid sequence of SEQ ID        NO:15.

In another embodiment, an antibody that specifically binds to Notch2 NRRis provided, wherein the antibody comprises:

-   -   (a) an HVR-H1 comprising the amino acid sequence of SEQ ID NO:2;    -   (b) an HVR-H2 comprising the amino acid sequence of SEQ ID NO:4;    -   (c) an HVR-H3 comprising the amino acid sequence of SEQ ID NO:5;    -   (d) an HVR-L1 comprising the amino acid sequence of SEQ ID NO:7;    -   (e) an HVR-L2 comprising the amino acid sequence of SEQ ID        NO:11; and    -   (f) an HVR-L3 comprising the amino acid sequence of SEQ ID        NO:16.

In another embodiment, an antibody that specifically binds to Notch2 NRRis provided, wherein the antibody comprises:

-   -   (a) an HVR-H1 comprising the amino acid sequence of SEQ ID NO:2;    -   (b) an HVR-H2 comprising the amino acid sequence of SEQ ID NO:4;    -   (c) an HVR-H3 comprising the amino acid sequence of SEQ ID NO:5;    -   (d) an HVR-L1 comprising the amino acid sequence of SEQ ID NO:8;    -   (e) an HVR-L2 comprising the amino acid sequence of SEQ ID        NO:12; and    -   (f) an HVR-L3 comprising the amino acid sequence of SEQ ID        NO:17.

In another embodiment, an antibody that specifically binds to Notch2 NRRis provided, wherein the antibody comprises:

-   -   (a) an HVR-H1 comprising the amino acid sequence of SEQ ID NO:2;    -   (b) an HVR-H2 comprising the amino acid sequence of SEQ ID NO:4;    -   (c) an HVR-H3 comprising the amino acid sequence of SEQ ID NO:5;    -   (d) an HVR-L1 comprising the amino acid sequence of SEQ ID NO:9;    -   (e) an HVR-L2 comprising the amino acid sequence of SEQ ID        NO:13; and    -   (f) an HVR-L3 comprising the amino acid sequence of SEQ ID        NO:18.

In certain embodiments, any of the above antibodies further comprises atleast one framework selected from a VH subgroup III consensus frameworkand a VL subgroup I consensus framework.

In certain embodiments, an anti-Notch2 NRR antibody is affinity matured.For example, any one or more of the following substitutions in theindicated HVR positions (Kabat numbered) may be made in any combination:

-   -   in HVR-H1 (SEQ ID NO:1): S28T; T30S;    -   in HVR-L1 (SEQ ID NO:6): S28N; 129N or V; S30R or K; S31R; Y32F    -   in HVR-L2 (SEQ ID NO:11): G50R; S53I or T; A55E    -   in HVR-L3 (SEQ ID NO:15): S93I or R; L96W or H        The specific antibodies disclosed herein, i.e., Antibody D as        well as affinity matured forms of Antibody D (D-1, D-2, and        D-3), may undergo further affinity maturation. Accordingly,        affinity matured forms of any of the antibodies described herein        are provided.

In certain embodiments, an anti-Notch2 NRR antibody having any of theabove HVR sequences can further comprise any suitable framework variabledomain sequence, provided binding activity to Notch2 NRR issubstantially retained. In certain embodiments, an anti-Notch2 NRRantibody comprises a human variable heavy (VH) consensus frameworksequence, as in any of the VH consensus framework sequences shown inFIGS. 16A and 16B. In one embodiment, the VH consensus frameworksequence comprises a human subgroup III heavy chain framework consensussequence, e.g., as shown in FIGS. 16A and 16B. In another embodiment,the VH consensus framework sequence comprises an “Acceptor 2” frameworksequence, e.g., as shown in FIGS. 16A and 16B. In a particularembodiment, the VH framework consensus sequence comprises FR1-FR4 ofAcceptor 2B or Acceptor 2D, wherein the FR4 comprises SEQ ID NO:35(FIGS. 16A and 16B), with the last residue of SEQ ID NO:35 (S11)optionally being substituted with alanine 1n a further particularembodiment, the VH framework consensus sequence comprises the sequencesof SEQ ID NOs:50; 51; 57 or 59; and 35, wherein S11 of SEQ ID NO:35 isoptionally substituted with alanine

In certain embodiments, an anti-Notch2 NRR antibody having any of theabove HVR sequences can further comprise a human variable light (VL)consensus framework sequence as shown in FIG. 17. In one embodiment, theVL consensus framework sequence comprises a human VL kappa subgroup Iconsensus framework (κv1) sequence, e.g., as shown in FIG. 17.

In another embodiment, the VL framework consensus sequence comprisesFR1-FR4 of huMAb4D5-8 as shown in FIG. 18 or 19. In a particularembodiment, the VL framework consensus sequence comprises the sequencesof SEQ ID NOs:60, 61, 62, and 63.

In another aspect, an anti-Notch2 NRR antibody comprises a heavy chainvariable domain (VH) sequence having at least 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an amino acidsequence selected from SEQ ID NOs:20-21. In certain embodiments, a VHsequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or99% identity contains substitutions (e.g., conservative substitutions),insertions, or deletions relative to the reference sequence, but ananti-Notch2 NRR antibody comprising that sequence retains the ability tobind to Notch2 NRR. In certain embodiments, a total of 1 to 10 aminoacids have been substituted, inserted and/or deleted in an amino acidsequence selected from SEQ ID NOs:20-21. In certain embodiments,substitutions, insertions, or deletions occur in regions outside theHVRs (i.e., in the FRs). In a particular embodiment, the VH comprisesone, two or three HVRs selected from: (a) an HVR-H1 comprising an aminoacid sequence that conforms to the consensus sequence of SEQ ID NO:3,(b) an HVR-H2 comprising the amino acid sequence of SEQ ID NO:4, and (c)an HVR-H3 comprising the amino acid sequence of SEQ ID NO:5. In one suchembodiment, HVR-H1 comprises an amino acid sequence selected from SEQ IDNOs:1-2.

In another aspect, an antibody that specifically binds to Notch2 NRR isprovided, wherein the antibody comprises a light chain variable domain(VL) having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%,or 100% sequence identity to an amino acid sequence selected from SEQ IDNOs:22-25. In certain embodiments, a VL sequence having at least 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity containssubstitutions (e.g., conservative substitutions), insertions, ordeletions relative to the reference sequence, but an anti-Notch2 NRRantibody comprising that sequence retains the ability to bind to Notch2NRR. In certain embodiments, a total of 1 to 10 amino acids have beensubstituted, inserted and/or deleted in an amino acid sequence selectedfrom SEQ ID NOs:22-25. In certain embodiments, the substitutions,insertions, or deletions occur in regions outside the HVRs (i.e., in theFRs). In a particular embodiment, the VL comprises one, two or threeHVRs selected from (a) an HVR-L1 comprising an amino acid sequence thatconforms to the consensus sequence of SEQ ID NO:10; (b) an HVR-L2comprising an amino acid sequence that conforms to the consensussequence of SEQ ID NO:14; and (c) an HVR-L3 comprising an amino acidsequence that conforms to the consensus sequence of SEQ ID NO:19. In onesuch embodiment, the VL comprises one, two or three HVRs selected from(a) an HVR-L1 comprising an amino acid sequence selected from SEQ IDNOs:6-9; (b) an HVR-L2 comprising an amino acid sequence selected fromSEQ ID NOs:11-13; and (c) an HVR-L3 comprising an amino acid sequenceselected from SEQ ID NOs:15-18. In one such embodiment, the VL comprisesone, two or three HVRs selected from (a) an HVR-L1 comprising the aminoacid sequence of SEQ ID NO:6; (b) an HVR-L2 comprising the amino acidsequence of SEQ ID NO:11; and (c) an HVR-L3 comprising the amino acidsequence of SEQ ID NO:15. In another such embodiment, the VL comprisesone, two or three HVRs selected from (a) an HVR-L1 comprising the aminoacid sequence of SEQ ID NO:7; (b) an HVR-L2 comprising the amino acidsequence of SEQ ID NO:11; and (c) an HVR-L3 comprising the amino acidsequence of SEQ ID NO:16. In another such embodiment, the VL comprisesone, two or three HVRs selected from (a) an HVR-L1 comprising the aminoacid sequence of SEQ ID NO:8; (b) an HVR-L2 comprising the amino acidsequence of SEQ ID NO:12; and (c) an HVR-L3 comprising the amino acidsequence of SEQ ID NO:17. In another such embodiment, the VL comprisesone, two or three HVRs selected from (a) an HVR-L1 comprising the aminoacid sequence of SEQ ID NO:9; (b) an HVR-L2 comprising the amino acidsequence of SEQ ID NO:13; and (c) an HVR-L3 comprising the amino acidsequence of SEQ ID NO:18.

In certain embodiments of the variant VH and VL sequences providedabove, substitutions, insertions, or deletions may occur within theHVRs. In such embodiments, substitutions, insertions, or deletions mayoccur within one or more HVRs so long as such alterations do notsubstantially reduce the ability of the antibody to bind antigen. Forexample, conservative alterations that do not substantially reducebinding affinity may be made in HVRs. In certain instances, alterationsin HVRs may actually improve antibody affinity. Such alterations may bemade in HVR “hotspots” (i.e., residues encoded by codons that undergomutation at high frequency during the somatic maturation process) inorder to increase antibody affinity. (See, e.g., Chowdhury, Methods Mol.Biol. 207:179-196, 2008.) In certain embodiments of the variant VH andVL sequences provided above, each HVR either is conserved (unaltered),or contains no more than a single amino acid substitution, insertion ordeletion.

In another aspect, an antibody that specifically binds Notch2 NRR isprovided, wherein the antibody comprises a VH as in any of theembodiments provided above, and a VL as in any of the embodimentsprovided above. In one embodiment, the antibody comprises a VH having atleast 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequenceidentity to the amino acid sequence of SEQ ID NO:20, and a VL having atleast 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequenceidentity to the amino acid sequence of SEQ ID NO:22. In one suchembodiment, the VH comprises one, two or three HVRs selected from: (a)an HVR-H1 comprising the amino acid sequence of SEQ ID NO:1, (b) anHVR-H2 comprising the amino acid sequence of SEQ ID NO:4, and (c) anHVR-H3 comprising the amino acid sequence of SEQ ID NO:5, and the VLcomprises one, two or three HVRs selected from (a) an HVR-L1 comprisingthe amino acid sequence of SEQ ID NO:6; (b) an HVR-L2 comprising theamino acid sequence of SEQ ID NO:11; and (c) an HVR-L3 comprising theamino acid sequence of SEQ ID NO:15. In a particular embodiment, theantibody comprises a VH comprising the amino acid sequence of SEQ IDNO:20, and a VL comprising the amino acid sequence of SEQ ID NO:22.

In another embodiment, an anti-Notch2 NRR antibody that specificallybinds Notch2 NRR comprises a VH having at least 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acidsequence of SEQ ID NO:21, and a VL having at least 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an amino acidsequence selected from SEQ ID NOs:23-25. In one such embodiment, the VHcomprises one, two or three HVRs selected from: (a) an HVR-H1 comprisingthe amino acid sequence of SEQ ID NO:2, (b) an HVR-H2 comprising theamino acid sequence of SEQ ID NO:4, and (c) an HVR-H3 comprising theamino acid sequence of SEQ ID NO:5, and the VL comprises one, two orthree HVRs selected from (a) an HVR-L1 comprising an amino acid sequenceselected from SEQ ID NOs:7-9; (b) an HVR-L2 comprising an amino acidsequence selected from SEQ ID NOs:11-13; and (c) an HVR-L3 comprising anamino acid sequence selected from SEQ ID NOs:16-18. In particularembodiments, the antibody comprises a VH comprising the amino acidsequence of SEQ ID NO:21 and a VL comprising an amino acid sequenceselected from SEQ ID NOs:23-25.

In certain embodiments, an affinity-matured form of any of the aboveantibodies is provided. In further embodiments, a recombinant proteinthat specifically binds Notch2 NRR is provided, wherein the recombinantprotein comprises an antigen binding site(s) of any of the aboveantibodies. In one such embodiment, a recombinant protein comprises anyone or more of the HVRs provided above.

In certain embodiments, a polynucleotide encoding any of the aboveantibodies is provided. In one embodiment, a vector comprising thepolynucleotide is provided. In one embodiment, a host cell comprisingthe vector is provided. In one embodiment, the host cell is eukaryotic.In one embodiment, the host cell is a CHO cell. In one embodiment, amethod of making an anti-Notch2 NRR antibody is provided, wherein themethod comprises culturing the host cell under conditions suitable forexpression of the polynucleotide encoding the antibody, and isolatingthe antibody.

In another embodiment, an isolated antibody is provided that binds tothe same epitope as an antibody provided herein. In one embodiment, anisolated anti-Notch2 NRR antibody is provided that binds to the sameepitope as an antibody selected from Antibody D, Antibody D-1, AntibodyD-2, and Antibody D-3. In another embodiment, the invention provides ananti-Notch2 NRR antibody that competes for binding with an antibodyselected from Antibody D, Antibody D-1, Antibody D-2, and Antibody D-3.In another embodiment, an isolated antibody is provided that binds to atleast one domain selected from the LNR-A domain and the HD-C domain ofNotch2. In one such embodiment, the antibody binds to both the LNR-Adomain and the HD-C domain. In another such embodiment, the antibodyfurther binds to the LNR-B and/or HD-N domains.

Any of the Notch2-specific antagonists provided herein may be used intherapeutic methods. In one aspect, a Notch2-specific antagonist for useas a medicament is provided. In further aspects, a Notch2-specificantagonist for use in treating a liver condition characterized by liverdamage is provided. In certain embodiments, a Notch2-specific antagonistfor use in a method of treatment is provided. In certain embodiments,the invention provides a Notch2-specific antagonist for use in a methodof treating an individual having a liver condition characterized byliver damage comprising administering to the individual an effectiveamount of the Notch2-specific antagonist. In one such embodiment, themethod further comprises administering to the individual an effectiveamount of at least one additional therapeutic agent, e.g., as describedbelow. An “individual” according to any of the above embodiments ispreferably a human.

In a further aspect, the invention provides for the use of aNotch2-specific antagonist in the manufacture or preparation of amedicament. In one embodiment, the medicament is for treatment of aliver condition characterized by liver damage. In a further embodiment,the medicament is for use in a method of treating a liver conditioncharacterized by liver damage comprising administering to an individualhaving a liver condition characterized by liver damage an effectiveamount of the medicament. In one such embodiment, the method furthercomprises administering to the individual an effective amount of atleast one additional therapeutic agent, e.g., as described below. An“individual” according to any of the above embodiments may be a human.

In a further aspect, the invention provides pharmaceutical formulationscomprising any of the Notch2-specific antagonists provided herein, e.g.,for use in any of the above therapeutic methods. In one embodiment, apharmaceutical formulation comprises any of the Notch2-specificantagonists provided herein and a pharmaceutically acceptable carrier.In another embodiment, a pharmaceutical formulation comprises any of theNotch2-specific antagonists provided herein and at least one additionaltherapeutic agent, e.g., as described below.

Antibodies of the invention can be used either alone or in combinationwith other agents in a therapy. For instance, an antibody of theinvention may be co-administered with at least one additionaltherapeutic agent.

Such combination therapies noted above encompass combined administration(where two or more therapeutic agents are included in the same orseparate formulations), and separate administration, in which case,administration of the antagonist of the invention can occur prior to,simultaneously, and/or following, administration of the additionaltherapeutic agent and/or adjuvant. Antagonists of the invention can alsobe used in combination with radiation therapy.

The antagonist can be administered to a human patient by any knownmethod, such as intravenous administration, e.g., as a bolus or bycontinuous infusion over a period of time, by intramuscular,intraperitoneal, intracerobrospinal, subcutaneous, intra-articular,intrasynovial, intrathecal, oral, topical, or inhalation routes. TheNotch2-specific antagonist might be administered as a protein or as anucleic acid encoding a protein (see, for example, WO96/07321). Othertherapeutic regimens may be combined with the administration of theNotch2-specific antagonist. The combined administration includesco-administration, using separate formulations or a singlepharmaceutical formulation, and consecutive administration in eitherorder, wherein preferably there is a time period while both (or all)active agents simultaneously exert their biological activities. In someembodiments, such combined therapy results in a synergistic therapeuticeffect.

The dosage and mode of administration will be chosen by the physicianaccording to known criteria. The appropriate dosage of antibody,oligopeptide or organic molecule will depend on the type of disease tobe treated, the severity and course of the disease, whether theantibody, oligopeptide or organic molecule is administered forpreventive or therapeutic purposes, previous therapy, the patient'sclinical history and response to the Notch2-specific antagonist, and thediscretion of the attending physician. The Notch2-specific antagonistcan be administered to the patient at one time or over a series oftreatments.

Success of treatment of liver disease can be monitored by assessingparameters of liver function and recovery. Such parameters include, butare not limited to, improved liver function tests, (e.g., assessingserum albumin, bilirubin, bile acids, total protein, clotting times),liver enzymes (e.g., alanine transaminase, aspartate transaminase,alkaline phosphatase, gamma glutamyl transpeptidase), histologicappearance (e.g., needle biopsy showing improved hepatic architecture),and imaging modalities (e.g., ultrasound, magnetic resonance imaging forfibrosis and liver size).

In a further aspect, an anti-Notch2 antibody used in any of the aboveembodiments may incorporate any of the features, singly or incombination, as described in Sections 1-7 below.

1. Antibody Affinity

In certain embodiments, an antibody provided herein has a dissociationconstant (Kd) of ≦1 μM, ≦100 nM, ≦10 nM, ≦1 nM, ≦0.1 nM, ≦0.01 nM, or≦0.001 nM (e.g., 10⁻⁸M or less, e.g. from 10⁻⁸M to 10⁻¹³M, e.g., from10⁻⁹M to 10⁻¹³ M). For example, the exemplary phage Antibody D-3 bindsto Notch2 with a Kd of 5 nM.

In one embodiment, Kd is measured by a radiolabeled antigen bindingassay (RIA) performed with the Fab version of an antibody of interestand its antigen as described by the following assay. Solution bindingaffinity of Fabs for antigen is measured by equilibrating Fab with aminimal concentration of (¹²⁵I)-labeled antigen in the presence of atitration series of unlabeled antigen, then capturing bound antigen withan anti-Fab antibody-coated plate (see, e.g., Chen et al., J. Mol. Biol.293:865-881 (1999)). To establish conditions for the assay, MICROTITER®multi-well plates (Thermo Scientific) are coated overnight with 5 μg/mlof a capturing anti-Fab antibody (Cappel Labs) in 50 mM sodium carbonate(pH 9.6), and subsequently blocked with 2% (w/v) bovine serum albumin inPBS for two to five hours at room temperature (approximately 23° C.). Ina non-adsorbent plate (Nunc #269620), 100 μM or 26 μM [¹²⁵I]-antigen aremixed with serial dilutions of a Fab of interest (e.g., consistent withassessment of the anti-VEGF antibody, Fab-12, in Presta et al., CancerRes. 57:4593-4599 (1997)). The Fab of interest is then incubatedovernight; however, the incubation may continue for a longer period(e.g., about 65 hours) to ensure that equilibrium is reached.Thereafter, the mixtures are transferred to the capture plate forincubation at room temperature (e.g., for one hour). The solution isthen removed and the plate washed eight times with 0.1% polysorbate 20(TWEEN-20®) in PBS. When the plates have dried, 150 μl/well ofscintillant (MICROSCINT-20™; Packard) is added, and the plates arecounted on a TOPCOUNT™ gamma counter (Packard) for ten minutes.Concentrations of each Fab that give less than or equal to 20% ofmaximal binding are chosen for use in competitive binding assays.

According to another embodiment, Kd is measured using surface plasmonresonance assays using a BIACORE®-2000 or a BIACORE®-3000 (BIAcore,Inc., Piscataway, N.J.) at 25° C. with immobilized antigen CM5 chips at˜10 response units (RU). Briefly, carboxymethylated dextran biosensorchips (CM5, BIACORE, Inc.) are activated withN-ethyl-N′-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) andN-hydroxysuccinimide (NHS) according to the supplier's instructions.Antigen is diluted with 10 mM sodium acetate, pH 4.8, to 5 μg/ml (˜0.2μM) before injection at a flow rate of 5 μl/minute to achieveapproximately 10 response units (RU) of coupled protein. Following theinjection of antigen, 1 M ethanolamine is injected to block unreactedgroups. For kinetics measurements, two-fold serial dilutions of Fab(0.78 nM to 500 nM) are injected in PBS with 0.05% polysorbate 20(TWEEN-20™) surfactant (PBST) at 25° C. at a flow rate of approximately25 μl/min. Association rates (k_(on)) and dissociation rates (k_(off))are calculated using a simple one-to-one Langmuir binding model(BIACORE® Evaluation Software version 3.2) by simultaneously fitting theassociation and dissociation sensorgrams. The equilibrium dissociationconstant (Kd) is calculated as the ratio k_(off)/k_(on). See, e.g., Chenet al., J. Mol. Biol. 293:865-881 (1999). If the on-rate exceeds 10⁶ M⁻¹s⁻¹ by the surface plasmon resonance assay above, then the on-rate canbe determined by using a fluorescent quenching technique that measuresthe increase or decrease in fluorescence emission intensity(excitation=295 nm; emission=340 nm, 16 nm band-pass) at 25° C. of a 20nM anti-antigen antibody (Fab form) in PBS, pH 7.2, in the presence ofincreasing concentrations of antigen as measured in a spectrometer, suchas a stop-flow equipped spectrophometer (Aviv Instruments) or a8000-series SLM-AMINCO™ spectrophotometer (ThermoSpectronic) with astirred cuvette.

2. Antibody Fragments

In certain embodiments, an antibody provided herein is an antibodyfragment. Antibody fragments include, but are not limited to, Fab, Fab′,Fab′-SH, F(ab′)₂, Fv, and scFv fragments, and other fragments describedbelow. For a review of certain antibody fragments, see Hudson et al.Nat. Med. 9:129-134 (2003). For a review of scFv fragments, see, e.g.,Pluckthün, in The Pharmacology of Monoclonal Antibodies, vol. 113,Rosenburg and Moore eds., (Springer-Verlag, New York), pp. 269-315(1994); see also WO 93/16185; and U.S. Pat. Nos. 5,571,894 and5,587,458. For discussion of Fab and F(ab′)₂ fragments comprisingsalvage receptor binding epitope residues and having increased in vivohalf-life, see U.S. Pat. No. 5,869,046.

Diabodies are antibody fragments with two antigen-binding sites that maybe bivalent or bispecific. See, for example, EP 404,097; WO 1993/01161;Hudson et al., Nat. Med. 9:129-134 (2003); and Hollinger et al., Proc.Natl. Acad. Sci. USA 90: 6444-6448 (1993). Triabodies and tetrabodiesare also described in Hudson et al., Nat. Med. 9:129-134 (2003).Single-domain antibodies are antibody fragments comprising all or aportion of the heavy chain variable domain or all or a portion of thelight chain variable domain of an antibody. In certain embodiments, asingle-domain antibody is a human single-domain antibody (Domantis,Inc., Waltham, Mass.; see, e.g., U.S. Pat. No. 6,248,516 B1).

Antibody fragments can be made by various techniques, including but notlimited to proteolytic digestion of an intact antibody as well asproduction by recombinant host cells (e.g. E. coli or phage).

3. Chimeric and Humanized Antibodies

In certain embodiments, an antibody provided herein is a chimericantibody. Certain chimeric antibodies are described, e.g., in U.S. Pat.No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA,81:6851-6855 (1984)). In one example, a chimeric antibody comprises anon-human variable region (e.g., a variable region derived from a mouse,rat, hamster, rabbit, or non-human primate, such as a monkey) and ahuman constant region. In a further example, a chimeric antibody is a“class switched” antibody in which the class or subclass has beenchanged from that of the parent antibody. Chimeric antibodies includeantigen-binding fragments thereof.

In certain embodiments, a chimeric antibody is a humanized antibody.Typically, a non-human antibody is humanized to reduce immunogenicity tohumans, while retaining the specificity and affinity of the parentalnon-human antibody. Generally, a humanized antibody comprises one ormore variable domains in which HVRs, e.g., CDRs, (or portions thereof)are derived from a non-human antibody, and FRs (or portions thereof) arederived from human antibody sequences. A humanized antibody optionallywill also comprise at least a portion of a human constant region. Insome embodiments, some FR residues in a humanized antibody aresubstituted with corresponding residues from a non-human antibody (e.g.,the antibody from which the HVR residues are derived), e.g., to restoreor improve antibody specificity or affinity.

Humanized antibodies and methods of making them are reviewed, e.g., inAlmagro and Fransson, Front. Biosci. 13:1619-1633 (2008), and arefurther described, e.g., in Riechmann et al., Nature 332:323-329 (1988);Queen et al., Proc. Nat'l Acad. Sci. USA 86:10029-10033 (1989); U.S.Pat. Nos. 5,821,337, 7,527,791, 6,982,321, and 7,087,409; Kashmiri etal., Methods 36:25-34 (2005) (describing SDR (a-CDR) grafting); Padlan,Mol. Immunol. 28:489-498 (1991) (describing “resurfacing”); Dall'Acquaet al., Methods 36:43-60 (2005) (describing “FR shuffling”); and Osbournet al., Methods 36:61-68 (2005) and Klimka et al., Br. J. Cancer,83:252-260 (2000) (describing the “guided selection” approach to FRshuffling).

Human framework regions that may be used for humanization include butare not limited to: framework regions selected using the “best-fit”method (see, e.g., Sims et al. J. Immunol. 151:2296 (1993)); frameworkregions derived from the consensus sequence of human antibodies of aparticular subgroup of light or heavy chain variable regions (see, e.g.,Carter et al. Proc. Natl. Acad. Sci. USA, 89:4285 (1992); and Presta etal. J. Immunol., 151:2623 (1993)); human mature (somatically mutated)framework regions or human germline framework regions (see, e.g.,Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008)); and frameworkregions derived from screening FR libraries (see, e.g., Baca et al., J.Biol. Chem. 272:10678-10684 (1997) and Rosok et al., J. Biol. Chem.271:22611-22618 (1996)).

4. Human Antibodies

In certain embodiments, an antibody provided herein is a human antibody.Human antibodies can be produced using various techniques known in theart. Human antibodies are described generally in van Dijk and van deWinkel, Curr. Opin. Pharmacol. 5: 368-74 (2001) and Lonberg, Curr. Opin.Immunol. 20:450-459 (2008).

Human antibodies may be prepared by administering an immunogen to atransgenic animal that has been modified to produce intact humanantibodies or intact antibodies with human variable regions in responseto antigenic challenge. Such animals typically contain all or a portionof the human immunoglobulin loci, which replace the endogenousimmunoglobulin loci, or which are present extrachromosomally orintegrated randomly into the animal's chromosomes. In such transgenicmice, the endogenous immunoglobulin loci have generally beeninactivated. For review of methods for obtaining human antibodies fromtransgenic animals, see Lonberg, Nat. Biotech. 23:1117-1125 (2005). Seealso, e.g., U.S. Pat. Nos. 6,075,181 and 6,150,584 describing XENOMOUSE™technology; U.S. Pat. No. 5,770,429 describing HUMAB® technology; U.S.Pat. No. 7,041,870 describing K-M MOUSE® technology, and U.S. PatentApplication Publication No. US 2007/0061900, describing VELOCIMOUSE®technology). Human variable regions from intact antibodies generated bysuch animals may be further modified, e.g., by combining with adifferent human constant region.

Human antibodies can also be made by hybridoma-based methods. Humanmyeloma and mouse-human heteromyeloma cell lines for the production ofhuman monoclonal antibodies have been described. (See, e.g., Kozbor J.Immunol., 133: 3001 (1984); Brodeur et al., Monoclonal AntibodyProduction Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc.,New York, 1987); and Boerner et al., J. Immunol., 147: 86 (1991).) Humanantibodies generated via human B-cell hybridoma technology are alsodescribed in Li et al., Proc. Natl. Acad. Sci. USA, 103:3557-3562(2006). Additional methods include those described, for example, in U.S.Pat. No. 7,189,826 (describing production of monoclonal human IgMantibodies from hybridoma cell lines) and Ni, Xiandai Mianyixue,26(4):265-268 (2006) (describing human-human hybridomas). Humanhybridoma technology (Trioma technology) is also described in Vollmersand Brandlein, Histology and Histopathology, 20(3):927-937 (2005) andVollmers and Brandlein, Methods and Findings in Experimental andClinical Pharmacology, 27(3):185-91 (2005).

Human antibodies may also be generated by isolating Fv clone variabledomain sequences selected from human-derived phage display libraries.Such variable domain sequences may then be combined with a desired humanconstant domain.

5. Library-Derived Antibodies

Antibodies of the invention may be isolated by screening combinatoriallibraries for antibodies with the desired activity or activities. Forexample, a variety of methods are known in the art for generating phagedisplay libraries and screening such libraries for antibodies possessingthe desired binding characteristics. Such methods are reviewed, e.g., inHoogenboom et al. in Methods in Molecular Biology 178:1-37 (O'Brien etal., ed., Human Press, Totowa, N.J., 2001) and further described, e.g.,in the McCafferty et al., Nature 348:552-554; Clackson et al., Nature352: 624-628 (1991); Marks et al., J. Mol. Biol. 222: 581-597 (1992);Marks and Bradbury, in Methods in Molecular Biology 248:161-175 (Lo,ed., Human Press, Totowa, N.J., 2003); Sidhu et al., J. Mol. Biol.338(2): 299-310 (2004); Lee et al., J. Mol. Biol. 340(5): 1073-1093(2004); Fellouse, Proc. Natl. Acad. Sci. USA 101(34): 12467-12472(2004); and Lee et al., J. Immunol. Methods 284(1-2): 119-132 (2004).

In certain phage display methods, repertoires of VH and VL genes areseparately cloned by polymerase chain reaction (PCR) and recombinedrandomly in phage libraries, which can then be screened forantigen-binding phage as described in Winter et al., Ann. Rev. Immunol.,12: 433-455 (1994). Phage typically display antibody fragments, eitheras single-chain Fv (scFv) fragments or as Fab fragments. Libraries fromimmunized sources provide high-affinity antibodies to the immunogenwithout the requirement of constructing hybridomas. Alternatively, thenaive repertoire can be cloned (e.g., from human) to provide a singlesource of antibodies to a wide range of non-self and also self antigenswithout any immunization as described by Griffiths et al., EMBO J, 12:725-734 (1993). Finally, naive libraries can also be made syntheticallyby cloning unrearranged V-gene segments from stem cells, and using PCRprimers containing random sequence to encode the highly variable CDR3regions and to accomplish rearrangement in vitro, as described byHoogenboom and Winter, J. Mol. Biol., 227: 381-388 (1992). Patentpublications describing human antibody phage libraries include, forexample: U.S. Pat. No. 5,750,373, and US Patent Publication Nos.2005/0079574, 2005/0119455, 2005/0266000, 2007/0117126, 2007/0160598,2007/0237764, 2007/0292936, and 2009/0002360.

Antibodies or antibody fragments isolated from human antibody librariesare considered human antibodies or human antibody fragments herein.

6. Multispecific Antibodies

In certain embodiments, an antibody provided herein is a multispecificantibody, e.g. a bispecific antibody. Multispecific antibodies aremonoclonal antibodies that have binding specificities for at least twodifferent sites. In certain embodiments, one of the bindingspecificities is for Notch2 and the other is for any other antigen. Incertain embodiments, bispecific antibodies may bind to two differentepitopes of Notch2. Bispecific antibodies may also be used to localizecytotoxic agents to cells which express Notch2. Bispecific antibodiescan be prepared as full length antibodies or antibody fragments.

Techniques for making multispecific antibodies include, but are notlimited to, recombinant co-expression of two immunoglobulin heavychain-light chain pairs having different specificities (see Milstein andCuello, Nature 305: 537 (1983)), WO 93/08829, and Traunecker et al.,EMBO J. 10: 3655 (1991)), and “knob-in-hole” engineering (see, e.g.,U.S. Pat. No. 5,731,168). Multi-specific antibodies may also be made byengineering electrostatic steering effects for making antibodyFc-heterodimeric molecules (WO 2009/089004A1); cross-linking two or moreantibodies or fragments (see, e.g., U.S. Pat. No. 4,676,980, and Brennanet al., Science, 229: 81 (1985)); using leucine zippers to producebi-specific antibodies (see, e.g., Kostelny et al., J. Immunol.,148(5):1547-1553 (1992)); using “diabody” technology for makingbispecific antibody fragments (see, e.g., Hollinger et al., Proc. Natl.Acad. Sci. USA, 90:6444-6448 (1993)); and using single-chain Fv (sFv)dimers (see, e.g. Gruber et al., J. Immunol., 152:5368 (1994)); andpreparing trispecific antibodies as described, e.g., in Tutt et al. J.Immunol. 147: 60 (1991).

Engineered antibodies with three or more functional antigen bindingsites, including “Octopus antibodies,” are also included herein (see,e.g. US 2006/0025576A1).

The antibody or fragment herein also includes a “Dual Acting FAb” or“DAF” comprising an antigen binding site that binds to Notch2 as well asanother, different antigen (see, US 2008/0069820, for example).

7. Antibody Variants

In certain embodiments, amino acid sequence variants of the antibodiesprovided herein are contemplated. For example, it may be desirable toimprove the binding affinity and/or other biological properties of theantibody. Amino acid sequence variants of an antibody may be prepared byintroducing appropriate modifications into the nucleotide sequenceencoding the antibody, or by peptide synthesis. Such modificationsinclude, for example, deletions from, and/or insertions into and/orsubstitutions of residues within the amino acid sequences of theantibody. Any combination of deletion, insertion, and substitution canbe made to arrive at the final construct, provided that the finalconstruct possesses the desired characteristics, e.g., antigen-binding.

a) Substitution, Insertion, and Deletion Variants

In certain embodiments, antibody variants having one or more amino acidsubstitutions are provided. Sites of interest for substitutionalmutagenesis include the HVRs and FRs. Conservative substitutions areshown in Table 1 under the heading of “conservative substitutions.” Moresubstantial changes are provided in Table 1 under the heading of“exemplary substitutions,” and as further described below in referenceto amino acid side chain classes. Amino acid substitutions may beintroduced into an antibody of interest and the products screened for adesired activity, e.g., retained/improved antigen binding, decreasedimmunogenicity, or improved ADCC or CDC.

Non-conservative substitutions will entail exchanging a member of one ofthese classes for another class.

One type of substitutional variant involves substituting one or morehypervariable region residues of a parent antibody (e.g. a humanized orhuman antibody). Generally, the resulting variant(s) selected forfurther study will have modifications (e.g., improvements) in certainbiological properties (e.g., increased affinity, reduced immunogenicity)relative to the parent antibody and/or will have substantially retainedcertain biological properties of the parent antibody. An exemplarysubstitutional variant is an affinity matured antibody, which may beconveniently generated, e.g., using phage display-based affinitymaturation techniques such as those described herein. Briefly, one ormore HVR residues are mutated and the variant antibodies displayed onphage and screened for a particular biological activity (e.g. bindingaffinity).

TABLE 1 Original Exemplary Preferred Residue Substitutions SubstitutionsAla (A) Val; Leu; Ile Val Arg (R) Lys; Gln; Asn Lys Asn (N) Gln; His;Asp, Lys; Arg Gln Asp (D) Glu; Asn Glu Cys (C) Ser; Ala Ser Gln (Q) Asn;Glu Asn Glu (E) Asp; Gln Asp Gly (G) Ala Ala His (H) Asn; Gln; Lys; ArgArg Ile (I) Leu; Val; Met; Ala; Phe; Norleucine Leu Leu (L) Norleucine;Ile; Val; Met; Ala; Phe Ile Lys (K) Arg; Gln; Asn Arg Met (M) Leu; Phe;Ile Leu Phe (F) Trp; Leu; Val; Ile; Ala; Tyr Tyr Pro (P) Ala Ala Ser (S)Thr Thr Thr (T) Val; Ser Ser Trp (W) Tyr; Phe Tyr Tyr (Y) Trp; Phe; Thr;Ser Phe Val (V) Ile; Leu; Met; Phe; Ala; Norleucine LeuAmino acids may be grouped according to common side-chain properties:

-   -   (1) hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile;    -   (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln;    -   (3) acidic: Asp, Glu;    -   (4) basic: H is, Lys, Arg;    -   (5) residues that influence chain orientation: Gly, Pro;    -   (6) aromatic: Trp, Tyr, Phe.

Alterations (e.g., substitutions) may be made in HVRs, e.g., to improveantibody affinity. Such alterations may be made in HVR “hotspots,” i.e.,residues encoded by codons that undergo mutation at high frequencyduring the somatic maturation process (see, e.g., Chowdhury, MethodsMol. Biol. 207:179-196 (2008)), and/or SDRs (a-CDRs), with the resultingvariant VH or VL being tested for binding affinity. Affinity maturationby constructing and reselecting from secondary libraries has beendescribed, e.g., in Hoogenboom et al. in Methods in Molecular Biology178:1-37 (O'Brien et al., ed., Human Press, Totowa, N.J., (2001).) Insome embodiments of affinity maturation, diversity is introduced intothe variable genes chosen for maturation by any of a variety of methods(e.g., error-prone PCR, chain shuffling, or oligonucleotide-directedmutagenesis). A secondary library is then created. The library is thenscreened to identify any antibody variants with the desired affinity.Another method to introduce diversity involves HVR-directed approaches,in which several HVR residues (e.g., 4-6 residues at a time) arerandomized. HVR residues involved in antigen binding may be specificallyidentified, e.g., using alanine scanning mutagenesis or modeling. CDR-H3and CDR-L3 in particular are often targeted.

In certain embodiments, substitutions, insertions, or deletions mayoccur within one or more HVRs so long as such alterations do notsubstantially reduce the ability of the antibody to bind antigen. Forexample, conservative alterations (e.g., conservative substitutions asprovided herein) that do not substantially reduce binding affinity maybe made in HVRs. Such alterations may be outside of HVR “hotspots” orSDRs. In certain embodiments of the variant VH and VL sequences providedabove, each HVR either is unaltered, or contains no more than one, twoor three amino acid substitutions.

A useful method for identification of residues or regions of an antibodythat may be targeted for mutagenesis is called “alanine scanningmutagenesis” as described by Cunningham and Wells (1989) Science,244:1081-1085. In this method, a residue or group of target residues(e.g., charged residues such as arg, asp, his, lys, and glu) areidentified and replaced by a neutral or negatively charged amino acid(e.g., alanine or polyalanine) to determine whether the interaction ofthe antibody with antigen is affected. Further substitutions may beintroduced at the amino acid locations demonstrating functionalsensitivity to the initial substitutions. Alternatively, oradditionally, a crystal structure of an antigen-antibody complex toidentify contact points between the antibody and antigen. Such contactresidues and neighboring residues may be targeted or eliminated ascandidates for substitution. Variants may be screened to determinewhether they contain the desired properties.

Amino acid sequence insertions include amino- and/or carboxyl-terminalfusions ranging in length from one residue to polypeptides containing ahundred or more residues, as well as intrasequence insertions of singleor multiple amino acid residues. Examples of terminal insertions includean antibody with an N-terminal methionyl residue. Other insertionalvariants of the antibody molecule include the fusion to the N- orC-terminus of the antibody to an enzyme (e.g. for ADEPT) or apolypeptide which increases the serum half-life of the antibody.

b) Glycosylation Variants

In certain embodiments, an antibody provided herein is altered toincrease or decrease the extent to which the antibody is glycosylated.Addition or deletion of glycosylation sites to an antibody may beconveniently accomplished by altering the amino acid sequence such thatone or more glycosylation sites is created or removed.

Where the antibody comprises an Fc region, the carbohydrate attachedthereto may be altered. Native antibodies produced by mammalian cellstypically comprise a branched, biantennary oligosaccharide that isgenerally attached by an N-linkage to Asn297 of the CH2 domain of the Fcregion. See, e.g., Wright et al. TIBTECH 15:26-32 (1997). Theoligosaccharide may include various carbohydrates, e.g., mannose,N-acetyl glucosamine (GlcNAc), galactose, and sialic acid, as well as afucose attached to a GlcNAc in the “stem” of the biantennaryoligosaccharide structure. In some embodiments, modifications of theoligosaccharide in an antibody of the invention may be made in order tocreate antibody variants with certain improved properties.

In one embodiment, antibody variants are provided having a carbohydratestructure that lacks fucose attached (directly or indirectly) to an Fcregion. For example, the amount of fucose in such antibody may be from1% to 80%, from 1% to 65%, from 5% to 65% or from 20% to 40%. The amountof fucose is determined by calculating the average amount of fucosewithin the sugar chain at Asn297, relative to the sum of allglycostructures attached to Asn 297 (e.g. complex, hybrid and highmannose structures) as measured by MALDI-TOF mass spectrometry, asdescribed in WO 2008/077546, for example. Asn297 refers to theasparagine residue located at about position 297 in the Fc region (Eunumbering of Fc region residues); however, Asn297 may also be locatedabout +3 amino acids upstream or downstream of position 297, i.e.,between positions 294 and 300, due to minor sequence variations inantibodies. Such fucosylation variants may have improved ADCC function.See, e.g., US Patent Publication Nos. US 2003/0157108 (Presta, L.); US2004/0093621 (Kyowa Hakko Kogyo Co., Ltd). Examples of publicationsrelated to “defucosylated” or “fucose-deficient” antibody variantsinclude: US 2003/0157108; WO 2000/61739; WO 2001/29246; US 2003/0115614;US 2002/0164328; US 2004/0093621; US 2004/0132140; US 2004/0110704; US2004/0110282; US 2004/0109865; WO 2003/085119; WO 2003/084570; WO2005/035586; WO 2005/035778; WO2005/053742; WO2002/031140; Okazaki etal. J. Mol. Biol. 336:1239-1249 (2004); Yamane-Ohnuki et al. Biotech.Bioeng. 87: 614 (2004). Examples of cell lines capable of producingdefucosylated antibodies include Lec13 CHO cells deficient in proteinfucosylation (Ripka et al. Arch. Biochem. Biophys. 249:533-545 (1986);US Pat Appl No US 2003/0157108 A1, Presta, L; and WO 2004/056312 A1,Adams et al., especially at Example 11), and knockout cell lines, suchas alpha-1,6-fucosyltransferase gene, FUT8, knockout CHO cells (see,e.g., Yamane-Ohnuki et al. Biotech. Bioeng. 87: 614 (2004); Kanda, Y. etal., Biotechnol. Bioeng., 94(4):680-688 (2006); and WO2003/085107).

Antibodies variants are further provided with bisected oligosaccharides,e.g., in which a biantennary oligosaccharide attached to the Fc regionof the antibody is bisected by GlcNAc. Such antibody variants may havereduced fucosylation and/or improved ADCC function. Examples of suchantibody variants are described, e.g., in WO 2003/011878 (Jean-Mairet etal.); U.S. Pat. No. 6,602,684 (Umana et al.); and US 2005/0123546 (Umanaet al.). Antibody variants with at least one galactose residue in theoligosaccharide attached to the Fc region are also provided. Suchantibody variants may have improved CDC function. Such antibody variantsare described, e.g., in WO 1997/30087 (Patel et al.); WO 1998/58964(Raju, S.); and WO 1999/22764 (Raju, S.).

c) Fc Region Variants

In certain embodiments, one or more amino acid modifications may beintroduced into the Fc region of an antibody provided herein, therebygenerating an Fc region variant. The Fc region variant may comprise ahuman Fc region sequence (e.g., a human IgG1, IgG2, IgG3 or IgG4 Fcregion) comprising an amino acid modification (e.g. a substitution) atone or more amino acid positions.

In certain embodiments, the invention contemplates an antibody variantthat possesses some but not all effector functions, which make it adesirable candidate for applications in which the half life of theantibody in vivo is important yet certain effector functions (such ascomplement and ADCC) are unnecessary or deleterious. In vitro and/or invivo cytotoxicity assays can be conducted to confirm thereduction/depletion of CDC and/or ADCC activities. For example, Fcreceptor (FcR) binding assays can be conducted to ensure that theantibody lacks FcγR binding (hence likely lacking ADCC activity), butretains FcRn binding ability. The primary cells for mediating ADCC, NKcells, express FcγRIII only, whereas monocytes express FcγRI, FcγRII andFcγRIII. FcR expression on hematopoietic cells is summarized in Table 3on page 464 of Ravetch and Kinet, Annu. Rev. Immunol. 9:457-492 (1991).Non-limiting examples of in vitro assays to assess ADCC activity of amolecule of interest is described in U.S. Pat. No. 5,500,362 (see, e.g.Hellstrom, I. et al. Proc. Nat'l Acad. Sci. USA 83:7059-7063 (1986)) andHellstrom, I et al., Proc. Nat'l Acad. Sci. USA 82:1499-1502 (1985);5,821,337 (see Bruggemann, M. et al., J. Exp. Med. 166:1351-1361(1987)). Alternatively, non-radioactive assays methods may be employed(see, for example, ACTI™ non-radioactive cytotoxicity assay for flowcytometry (CellTechnology, Inc. Mountain View, Calif.; and CytoTox 96®non-radioactive cytotoxicity assay (Promega, Madison, Wis.). Usefuleffector cells for such assays include peripheral blood mononuclearcells (PBMC) and Natural Killer (NK) cells. Alternatively, oradditionally, ADCC activity of the molecule of interest may be assessedin vivo, e.g., in a animal model such as that disclosed in Clynes et al.Proc. Nat'l Acad. Sci. USA 95:652-656 (1998). C1q binding assays mayalso be carried out to confirm that the antibody is unable to bind C1qand hence lacks CDC activity. See, e.g., C1q and C3c binding ELISA in WO2006/029879 and WO 2005/100402. To assess complement activation, a CDCassay may be performed (see, for example, Gazzano-Santoro et al., J.Immunol. Methods 202:163 (1996); Cragg, M. S. et al., Blood101:1045-1052 (2003); and Cragg, M. S. and M. J. Glennie, Blood103:2738-2743 (2004)). FcRn binding and in vivo clearance/half lifedeterminations can also be performed using methods known in the art(see, e.g., Petkova, S. B. et al., Intl Immunol. 18(12):1759-1769(2006)).

Antibodies with reduced effector function include those withsubstitution of one or more of Fc region residues 238, 265, 269, 270,297, 327 and 329 (U.S. Pat. No. 6,737,056). Such Fc mutants include Fcmutants with substitutions at two or more of amino acid positions 265,269, 270, 297 and 327, including the so-called “DANA” Fc mutant withsubstitution of residues 265 and 297 to alanine (U.S. Pat. No.7,332,581).

Certain antibody variants with improved or diminished binding to FcRsare described. (See, e.g., U.S. Pat. No. 6,737,056; WO 2004/056312, andShields et al., J. Biol. Chem. 9(2): 6591-6604 (2001).)

In certain embodiments, an antibody variant comprises an Fc region withone or more amino acid substitutions which improve ADCC, e.g.,substitutions at positions 298, 333, and/or 334 of the Fc region (EUnumbering of residues).

In some embodiments, alterations are made in the Fc region that resultin altered (i.e., either improved or diminished) C1q binding and/orComplement Dependent Cytotoxicity (CDC), e.g., as described in U.S. Pat.No. 6,194,551, WO 99/51642, and Idusogie et al. J. Immunol. 164:4178-4184 (2000).

Antibodies with increased half lives and improved binding to theneonatal Fc receptor (FcRn), which is responsible for the transfer ofmaternal IgGs to the fetus (Guyer et al., J. Immunol. 117:587 (1976) andKim et al., J. Immunol. 24:249 (1994)), are described inUS2005/0014934A1 (Hinton et al.). Those antibodies comprise an Fc regionwith one or more substitutions therein which improve binding of the Fcregion to FcRn. Such Fc variants include those with substitutions at oneor more of Fc region residues: 238, 256, 265, 272, 286, 303, 305, 307,311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, 413, 424 or 434,e.g., substitution of Fc region residue 434 (U.S. Pat. No. 7,371,826).

See also Duncan & Winter, Nature 322:738-40 (1988); U.S. Pat. No.5,648,260; U.S. Pat. No. 5,624,821; and WO 94/29351 concerning otherexamples of Fc region variants.

d) Cysteine Engineered Antibody Variants

In certain embodiments, it may be desirable to create cysteineengineered antibodies, e.g., “thioMAbs,” in which one or more residuesof an antibody are substituted with cysteine residues. In particularembodiments, the substituted residues occur at accessible sites of theantibody. By substituting those residues with cysteine, reactive thiolgroups are thereby positioned at accessible sites of the antibody andmay be used to conjugate the antibody to other moieties, such as drugmoieties or linker-drug moieties, to create an immunoconjugate, asdescribed further herein. In certain embodiments, any one or more of thefollowing residues may be substituted with cysteine: V205 (Kabatnumbering) of the light chain; A118 (EU numbering) of the heavy chain;and S400 (EU numbering) of the heavy chain Fc region. Cysteineengineered antibodies may be generated as described, e.g., in U.S. Pat.No. 7,521,541.

e) Antibody Derivatives

In certain embodiments, an antibody provided herein may be furthermodified to contain additional nonproteinaceous moieties that are knownin the art and readily available. The moieties suitable forderivatization of the antibody include but are not limited to watersoluble polymers. Non-limiting examples of water soluble polymersinclude, but are not limited to, polyethylene glycol (PEG), copolymersof ethylene glycol/propylene glycol, carboxymethylcellulose, dextran,polyvinyl alcohol, polyvinyl pyrrolidone, poly-1,3-dioxolane,poly-1,3,6-trioxane, ethylene/maleic anhydride copolymer, polyaminoacids(either homopolymers or random copolymers), and dextran or poly(n-vinylpyrrolidone)polyethylene glycol, propropylene glycol homopolymers,prolypropylene oxide/ethylene oxide co-polymers, polyoxyethylatedpolyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof.Polyethylene glycol propionaldehyde may have advantages in manufacturingdue to its stability in water. The polymer may be of any molecularweight, and may be branched or unbranched. The number of polymersattached to the antibody may vary, and if more than one polymer areattached, they can be the same or different molecules. In general, thenumber and/or type of polymers used for derivatization can be determinedbased on considerations including, but not limited to, the particularproperties or functions of the antibody to be improved, whether theantibody derivative will be used in a therapy under defined conditions,etc.

In another embodiment, conjugates of an antibody and nonproteinaceousmoiety that may be selectively heated by exposure to radiation areprovided. In one embodiment, the nonproteinaceous moiety is a carbonnanotube (Kam et al., Proc. Natl. Acad. Sci. USA 102: 11600-11605(2005)). The radiation may be of any wavelength, and includes, but isnot limited to, wavelengths that do not harm ordinary cells, but whichheat the nonproteinaceous moiety to a temperature at which cellsproximal to the antibody-nonproteinaceous moiety are killed.

B. Recombinant Methods and Compositions

Antibodies may be produced using recombinant methods and compositions,e.g., as described in U.S. Pat. No. 4,816,567. In one embodiment,isolated nucleic acid encoding an anti-Notch2 antibody described hereinis provided. Such nucleic acid may encode an amino acid sequencecomprising the VL and/or an amino acid sequence comprising the VH of theantibody (e.g., the light and/or heavy chains of the antibody). In afurther embodiment, one or more vectors (e.g., expression vectors)comprising such nucleic acid are provided. In a further embodiment, ahost cell comprising such nucleic acid is provided. In one suchembodiment, a host cell comprises (e.g., has been transformed with): (1)a vector comprising a nucleic acid that encodes an amino acid sequencecomprising the VL of the antibody and an amino acid sequence comprisingthe VH of the antibody, or (2) a first vector comprising a nucleic acidthat encodes an amino acid sequence comprising the VL of the antibodyand a second vector comprising a nucleic acid that encodes an amino acidsequence comprising the VH of the antibody. In one embodiment, the hostcell is eukaryotic, e.g. a Chinese Hamster Ovary (CHO) cell or lymphoidcell (e.g., Y0, NS0, Sp20 cell). In one embodiment, a method of makingan anti-Notch2 antibody is provided, wherein the method comprisesculturing a host cell comprising a nucleic acid encoding the antibody,as provided above, under conditions suitable for expression of theantibody, and optionally recovering the antibody from the host cell (orhost cell culture medium).

For recombinant production of an anti-Notch2 antibody, nucleic acidencoding an antibody, e.g., as described above, is isolated and insertedinto one or more vectors for further cloning and/or expression in a hostcell. Such nucleic acid may be readily isolated and sequenced usingconventional procedures (e.g., by using oligonucleotide probes that arecapable of binding specifically to genes encoding the heavy and lightchains of the antibody).

Suitable host cells for cloning or expression of antibody-encodingvectors include prokaryotic or eukaryotic cells described herein. Forexample, antibodies may be produced in bacteria, in particular whenglycosylation and Fc effector function are not needed. For expression ofantibody fragments and polypeptides in bacteria, see, e.g., U.S. Pat.Nos. 5,648,237, 5,789,199, and 5,840,523. (See also Charlton, Methods inMolecular Biology, Vol. 248 (B. K. C. Lo, ed., Humana Press, Totowa,N.J., 2003), pp. 245-254, describing expression of antibody fragments inE. coli.) After expression, the antibody may be isolated from thebacterial cell paste in a soluble fraction and can be further purified.

In addition to prokaryotes, eukaryotic microbes such as filamentousfungi or yeast are suitable cloning or expression hosts forantibody-encoding vectors, including fungi and yeast strains whoseglycosylation pathways have been “humanized,” resulting in theproduction of an antibody with a partially or fully human glycosylationpattern. See Gerngross, Nat. Biotech. 22:1409-1414 (2004), and Li etal., Nat. Biotech. 24:210-215 (2006).

Suitable host cells for the expression of glycosylated antibody are alsoderived from multicellular organisms (invertebrates and vertebrates).Examples of invertebrate cells include plant and insect cells. Numerousbaculoviral strains have been identified which may be used inconjunction with insect cells, particularly for transfection ofSpodoptera frugiperda cells.

Plant cell cultures can also be utilized as hosts. See, e.g., U.S. Pat.Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429(describing PLANTIBODIES™ technology for producing antibodies intransgenic plants).

Vertebrate cells may also be used as hosts. For example, mammalian celllines that are adapted to grow in suspension may be useful. Otherexamples of useful mammalian host cell lines are monkey kidney CV1 linetransformed by SV40 (COS-7); human embryonic kidney line (293 or 293cells as described, e.g., in Graham et al., J. Gen Virol. 36:59 (1977));baby hamster kidney cells (BHK); mouse sertoli cells (TM4 cells asdescribed, e.g., in Mather, Biol. Reprod. 23:243-251 (1980)); monkeykidney cells (CV1); African green monkey kidney cells (VERO-76); humancervical carcinoma cells (HELA); canine kidney cells (MDCK; buffalo ratliver cells (BRL 3A); human lung cells (W138); human liver cells (HepG2); mouse mammary tumor (MMT 060562); TR1 cells, as described, e.g., inMather et al., Annals N.Y. Acad. Sci. 383:44-68 (1982); MRC 5 cells; andFS4 cells. Other useful mammalian host cell lines include Chinesehamster ovary (CHO) cells, including DHFR⁻ CHO cells (Urlaub et al.,Proc. Natl. Acad. Sci. USA 77:4216 (1980)); and myeloma cell lines suchas Y0, NS0 and Sp2/0. For a review of certain mammalian host cell linessuitable for antibody production, see, e.g., Yazaki and Wu, Methods inMolecular Biology, Vol. 248 (B. K. C. Lo, ed., Humana Press, Totowa,N.J.), pp. 255-268 (2003).

C. Assays

Anti-Notch2 antibodies provided herein may be identified, screened for,or characterized for their physical/chemical properties and/orbiological activities by various assays known in the art.

1. Binding Assays and Other Assays

In one aspect, an antibody of the invention is tested for its antigenbinding activity, e.g., by known methods such as ELISA, Western blot,etc.

In another aspect, competition assays may be used to identify anantibody that competes with Antibody D, Antibody D-1, Antibody D-2, orAntibody D-3 for binding to Notch2. In certain embodiments, such acompeting antibody binds to the same epitope (e.g., a linear or aconformational epitope) that is bound by Antibody D, Antibody D-1,Antibody D-2, or Antibody D-3. Detailed exemplary methods for mapping anepitope to which an antibody binds are provided in Morris (1996)“Epitope Mapping Protocols,” in Methods in Molecular Biology vol. 66(Humana Press, Totowa, N.J.).

In an exemplary competition assay, immobilized Notch2 is incubated in asolution comprising a first labeled antibody that binds to Notch2 (e.g.,Antibody D, Antibody D-1, Antibody D-2, or Antibody D-3) and a secondunlabeled antibody that is being tested for its ability to compete withthe first antibody for binding to Notch2. The second antibody may bepresent in a hybridoma supernatant. As a control, immobilized Notch2 isincubated in a solution comprising the first labeled antibody but notthe second unlabeled antibody. After incubation under conditionspermissive for binding of the first antibody to Notch2, excess unboundantibody is removed, and the amount of label associated with immobilizedNotch2 is measured. If the amount of label associated with immobilizedNotch2 is substantially reduced in the test sample relative to thecontrol sample, then that indicates that the second antibody iscompeting with the first antibody for binding to Notch2. See Harlow andLane (1988) Antibodies: A Laboratory Manual ch. 14 (Cold Spring HarborLaboratory, Cold Spring Harbor, N.Y.).

2. Activity Assays

In one aspect, assays are provided for identifying anti-Notch2antibodies thereof having biological activity. Biological activity mayinclude, e.g., inhibition or reduction of Notch2 activity, e.g., Notch2signaling. Antibodies having such biological activity in vivo and/or invitro are also provided.

In certain embodiments, an anti-Notch2 NRR antibody of the invention istested for its ability to inhibit generation of marginal zone B cells.An exemplary assay is provided in the Examples. In certain otherembodiments, an antibody of the invention is tested for its ability toinhibit expression of a reporter gene that is responsive to Notch2signaling.

D. Immunoconjugates

The invention also provides immunoconjugates comprising an anti-Notch2antibody herein conjugated to one or more cytotoxic agents, such aschemotherapeutic agents or drugs, growth inhibitory agents, toxins(e.g., protein toxins, enzymatically active toxins of bacterial, fungal,plant, or animal origin, or fragments thereof), or radioactive isotopes.

In one embodiment, an immunoconjugate is an antibody-drug conjugate(ADC) in which an antibody is conjugated to one or more drugs, includingbut not limited to a maytansinoid (see U.S. Pat. Nos. 5,208,020,5,416,064 and European Patent EP 0 425 235 B1); an auristatin such asmonomethylauristatin drug moieties DE and DF (MMAE and MMAF) (see U.S.Pat. Nos. 5,635,483 and 5,780,588, and 7,498,298); a dolastatin; acalicheamicin or derivative thereof (see U.S. Pat. Nos. 5,712,374,5,714,586, 5,739,116, 5,767,285, 5,770,701, 5,770,710, 5,773,001, and5,877,296; Hinman et al., Cancer Res. 53:3336-3342 (1993); and Lode etal., Cancer Res. 58:2925-2928 (1998)); an anthracycline such asdaunomycin or doxorubicin (see Kratz et al., Current Med. Chem.13:477-523 (2006); Jeffrey et al., Bioorganic & Med. Chem. Letters16:358-362 (2006); Torgov et al., Bioconj. Chem. 16:717-721 (2005); Nagyet al., Proc. Natl. Acad. Sci. USA 97:829-834 (2000); Dubowchik et al.,Bioorg. & Med. Chem. Letters 12:1529-1532 (2002); King et al., J. Med.Chem. 45:4336-4343 (2002); and U.S. Pat. No. 6,630,579); methotrexate;vindesine; a taxane such as docetaxel, paclitaxel, larotaxel, tesetaxel,and ortataxel; a trichothecene; and CC1065.

In another embodiment, an immunoconjugate comprises an antibody asdescribed herein conjugated to an enzymatically active toxin or fragmentthereof, including but not limited to diphtheria A chain, nonbindingactive fragments of diphtheria toxin, exotoxin A chain (from Pseudomonasaeruginosa), ricin A chain, abrin A chain, modeccin A chain,alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolacaamericana proteins (PAPI, PAPII, and PAP-S), momordica charantiainhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin,mitogellin, restrictocin, phenomycin, enomycin, and the tricothecenes.

In another embodiment, an immunoconjugate comprises an antibody asdescribed herein conjugated to a radioactive atom to form aradioconjugate. A variety of radioactive isotopes are available for theproduction of radioconjugates. Examples include At²¹¹, I¹³¹, I¹²⁵, Y⁹⁰,Re¹⁸⁶, Re¹⁸⁸, Sm¹⁵³, Bi²¹², P³², Pb²¹² and radioactive isotopes of Lu.When the radioconjugate is used for detection, it may comprise aradioactive atom for scintigraphic studies, for example tc99m or I123,or a spin label for nuclear magnetic resonance (NMR) imaging (also knownas magnetic resonance imaging, mri), such as iodine-123 again,iodine-131, indium-111, fluorine-19, carbon-13, nitrogen-15, oxygen-17,gadolinium, manganese or iron.

Conjugates of an antibody and cytotoxic agent may be made using avariety of bifunctional protein coupling agents such asN-succinimidyl-3-(2-pyridyldithio) propionate (SPDP),succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC),iminothiolane (IT), bifunctional derivatives of imidoesters (such asdimethyl adipimidate HCl), active esters (such as disuccinimidylsuberate), aldehydes (such as glutaraldehyde), bis-azido compounds (suchas bis (p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (suchas bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such astoluene 2,6-diisocyanate), and bis-active fluorine compounds (such as1,5-difluoro-2,4-dinitrobenzene). For example, a ricin immunotoxin canbe prepared as described in Vitetta et al., Science 238:1098 (1987).Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent forconjugation of radionucleotide to the antibody. See WO94/11026. Thelinker may be a “cleavable linker” facilitating release of a cytotoxicdrug in the cell. For example, an acid-labile linker,peptidase-sensitive linker, photolabile linker, dimethyl linker ordisulfide-containing linker (Chari et al., Cancer Res. 52:127-131(1992); U.S. Pat. No. 5,208,020) may be used.

The immunuoconjugates or ADCs herein expressly contemplate, but are notlimited to such conjugates prepared with cross-linker reagentsincluding, but not limited to, BMPS, EMCS, GMBS, HBVS, LC-SMCC, MBS,MPBH, SBAP, SIA, SIAB, SMCC, SMPB, SMPH, sulfo-EMCS, sulfo-GMBS,sulfo-KMUS, sulfo-MBS, sulfo-SIAB, sulfo-SMCC, and sulfo-SMPB, and SVSB(succinimidyl-(4-vinylsulfone)benzoate) which are commercially available(e.g., from Pierce Biotechnology, Inc., Rockford, Ill., U.S.A).

III. Examples Example 1 Isolation and Transcriptional Profiling of LiverProgenitor Cells

To identify the signals that regulate hepatocyte differentiation fromliver progenitor cells, transcriptional profiling was performed on adultliver progenitors (oval cells) from mice fed a choline deficient,ethionine supplemented (CDE) diet (FIG. 1A; FIG. 5A). This CDE model isknown in the art as a model of chronic liver disease. CDE is also asteatohepatitis model (NASH). Chronic CDE can also lead tohepatocellular carcinoma (HCC), thereby also serving as a model for HCC.To induce an oval cell response, 8-12 week-old female C57BL/6N mice(Charles River) were fed a choline deficient diet (20% Lard; TekladTD.04523) supplemented with 0.15% (w/v) Ethionine in the drinking water(Akhurst et al., Hepatology 34(3):519 (2001)). Liver non-parenchymalcells were isolated according to the protocol of del Castillo (delCastillo et al., Am. J. Pathol., 172(5):1238 (2008)) with the additionof 0.04% Hyaluronidase (Sigma) to the in vitro dissociation step.

Epithelial Cell Adhesion Molecule (EpCAM)-expressing progenitor cellsand normal bile duct cells from livers of CDE-fed and control mice wereisolated by fluorescence activated cell sorting (FACS; FIG. 4A-B).C57BL/6 mouse livers were perfused with a Collagenase/Pronase solutionand the liver was dissociated and further incubated in the presence ofDNase and Hyaluronidase (FIG. 4B). The resulting cell suspension waspassed over a 30%/70% Percoll density gradient by centrifugation at 2500RPM for 30 minutes. Cells from the 30%/70% Percoll interface, consistingmostly of non-parenchymal cells, were stained with fluorescently labeledantibodies to EpCAM (BioLegend) and CD45 (BD Pharmingen). Flow cytometrywas used to isolate EpCAM⁺/CD45⁻ cells from mice fed CDE or standardrodent diet. The majority of EpCAM⁺/CD45⁻ cells from CDE-fed mice wereoval cells, while the majority of EpCAM⁺/CD45⁻ cells from standarddiet-fed mice were bile duct cells. QRT-PCR analysis on RNA from FACSsorted cells confirmed that EpCAM⁺/CD45⁻-sorted cells were greatlyenriched for EpCAM as well as CK19 (FIG. 4B), indicating a successfulpositive selection of EpCAM⁺ progenitor and bile duct cells.

In addition to isolating progenitor cells by FACS sorting, progenitorand normal bile duct cells were isolated by laser capturemicrodissection (LCM) from hematoxylin and eosin (H&E) stained liversections. Livers from C57BL/6N 8-12 week-old female mice fed normal chowor CDE diet were removed and immediately flash frozen in liquidNitrogen. Flash frozen liver pieces were placed in prechilled plasticmolds, embedded in TISSUE-TEK OCT Compound (Sakura, The Netherlands) andimmediately placed on a dry ice/2-methylbutane bath until frozen. Theembedded frozen liver pieces were cut into 7-8 μm sections at −14° C.,adhered to metal frame membrane slides (MMI, Eching, Germany), fixed,stained with hematoxylin and eosin, and dried. Laser microdissection wasused to isolate 1-2 mm² of oval cell or normal bile duct tissue persample.

RNA from flow-sorted and laser microdissected tissue was isolated usingthe RNEASY Micro Kit (Qiagen). For microarray analysis RNA fromflow-sorted and laser microdissected tissue, as well as whole livercontrols, spiked with Agilent RNA Spike-In RNA (Agilent), was submittedto two rounds of amplification with Message AmpII (Ambion) andhybridized to Whole Mouse Genome Oligo 44k microarrays. Log expressionratios were exported and analyzed using Partek Genomic Suite (Partek).Quantitative real-time PCR (QRTPCR) was performed using the TAQMANOne-Step RT-PCR Kit for one step reactions using the 7900 HT RT-PCRsystem (Applied Biosystems) with TAQMAN probes (Applied Biosystems) orHigh Capacity cDNA RT kit with TAQMAN Fast Advanced Master Mix using theViia7 RT-PCR system (Applied Biosystems) with custom designed lowdensity arrays.

Enrichment of bile duct and progenitor cell-associated transcripts, suchas EpCAM and Keratin19, confirmed effective isolation of bile duct andprogenitor cells (FIG. 1E). Using supervised analysis (FIG. 5B), genesexpressed more highly in liver progenitor cells than in closely relatednormal bile duct cells were identified (Table 2). This supervisedanalysis correlated highly with progenitor cell associated transcriptsidentified by unbiased principle components analysis (FIG. 5C-H). Theexpression pattern of these genes was validated on independent samplesby QRT-PCR (FIG. 6) and immunofluorescence, which confirmed that membersof the Notch signaling pathway, including Jag1 and Notch2, as well asHes1 and Hey1, among other target genes, were upregulated in liverprogenitor cells compared to bile duct cells (FIG. 1C-G). A selection ofmicroarray probes for Notch pathway-associated transcripts distinguishedflow-sorted oval cells from flow-sorted biliary cells and whole liver bysupervised clustering (FIG. 1B). Supervised hierarchical clustering of29 Notch pathway-associated genes, including receptors, ligands,transcription factors, and select target genes, differentiated wholeliver, bile ducts, and oval cells into separate clusters (Rand index=1,3 clusters; Rand index=0.7607, 3 clusters, for randomly generated listof 29 genes) and revealed differences between normal bile duct cells andprogenitors. The data showed significant upregulation of Jag1 (p=0.0006;FIG. 1C), Notch2 (p=0.0006; FIG. 1D), Hes1 (p=0.0035; FIG. 1E), and Hey1(p=0.0087; FIG. 1F) in liver progenitor cells. Immunofluorescencestaining confirmed that Jag1 was more highly expressed in EpCAM⁺ ovalcells radiating out from the portal vein in a CDE liver than in adjacentnormal bile duct cells. Immunhistochemistry for Hes1 confirmed that Hes1positive cells were largely confined to the oval cell and bile ductcompartments, with some staining in other non-parenchymal cells of theliver (FIG. 1G).

TABLE 2 Oval Cell Associated Genes log2 (Fold UNQ_Short_Name ProbeIDt-statistic pvalue Change) ADAMTS9 A_52_P49321 5.272 0 2.132 ANXA9A_51_P451482 6.839 0 2.578 APP A_52_P381311 5.045 0 2.531 BMP8BA_51_P411926 6.273 0 3.562 CHRNB1 A_51_P475342 5.32 0 1.912 CTGFA_51_P157042 7.146 0 3.336 DTNA A_52_P108607 5.585 0 3.558 EmbiginA_51_P382849 6.196 0 3.246 Epdr2 A_52_P577388 5.703 0 2.431 EPHA7A_52_P504787 5.855 0 2.255 FADS3 A_52_P451796 5.809 0 2.163 Foxc1A_51_P107686 5.098 0 2.77 GSPT1 A_52_P354785 5.672 0 2.726 Hig2IA_52_P321150 5.522 0 1.696 ID2 A_52_P240542 5.054 0 1.504 Ifrd1A_51_P367060 5.888 0 1.609 JAG1 A_52_P634090 5.368 0 2.377 LTBA_51_P302358 6.15 0 2.428 MAL A_52_P562661 5.619 0 3.02 Mex3aA_52_P706060 5.228 0 3.582 MFI2 A_51_P324351 7.735 0 3.397 MYCA_51_P102096 5.498 0 1.522 NFAM1 A_52_P686701 5.472 0 2.09 NFKB1A_52_P32733 5.348 0 2.398 Nrarp A_51_P504354 5.261 0 2.585 peg3A_51_P206037 6.329 0 2.84 RASL11A A_51_P340699 5.425 0 2.303 SLIT2A_51_P496569 5.964 0 3.261 SPATA7 A_52_P134680 5.187 0 1.926 SPRR1AA_51_P139678 7.825 0 2.8 TNFAIP8 A_51_P435968 6.322 0 2.403 TNFRSF12AA_51_P131408 6.641 0 2.397 tp53 A_52_P957260 5.399 0 2.388 TRIM47A_51_P437176 5.633 0 1.592 Trio A_51_P319662 6.257 0 2.81 TTYH1A_52_P475052 5.668 0 2.442 USP47 A_52_P610967 5.021 0 2.018 VCAM1A_51_P210956 5.696 0 1.847

Example 2 Identification of Expression Patterns of Oval Cell-AssociatedGenes

The microarray data from RNA isolated from oval and bile duct cellsisolated FACS (Flow) or microdisection (LCM) and control and CDE liverswere examined for the expression of putative markers of oval cells andmarkers of other select cell types. Albumin transcript was detected atrelatively high levels in all groups (FIG. 6A-1), whereas AFP, a markerof immature hepatocytes that is upregulated during chronic liver damage,was greatly enriched in LCM oval cells, but virtually absent from theother cell types examined (FIG. 6A-1). To determine why AFP was presentonly in LCM oval cells, immunofluorescence was performed on 7 μm frozenCDE liver sections that had been briefly air-dried and fixed in 4%paraformaldehyde in PBS. After blocking with normal horse serum in PBS,sections were incubated with fluorescently labeled antibodies to EpCAM(BioLegend), Sca1 (BD Pharmingen), CD90 (BioLegend), or with unlabeledprimary antibodies to AFP (R&D Systems) and CK19 (Santa Cruz Biotech)followed by incubation with fluorescently labeled secondary antibodies(Invitrogen). AFP was expressed in only a subset of hepatocytes, oftenin close proximity to EpCAM⁺ oval cells. However, AFP expression couldnot be observed in oval cells themselves.

LCM isolates expressed high levels of the myofibroblast marker SMA andthe mesenchymal cell marker CD90/Thy1 (FIG. 6A-3), possibly as a resultof inclusion of periportal myofibroblasts, which are positive for bothCD90/Thy1 and SMA. Thus, it appears that the LCM samples contain aheterogeneous mixture of cell types that at least includemyofibroblasts, and in the case of LCM samples from CDE livers, AFPpositive hepatocytes adjacent to cords of microdissected oval cells.Though Sca1 also marks mesenchymal cells, it also appears to beexpressed in bile duct cells and oval cells themselves, as thetranscript is found at high levels in both the FACS-sorted and LCMsamples (FIG. 6A-2). The oval cell markers CD13, Sox9, FoxL, and FoxJ1were also expressed in both FACS-sorted and LCM samples. Except forSox9, which was more highly expressed in FACS oval cells, each of thesemarkers was expressed at comparable levels in CDE oval cells and innormal bile ducts. Expression patterns of oval cell-associated genes andgenes marking other hepatic cell types were confirmed in independentsamples by QRT-PCR. For these experiments, CDE oval cells were enrichedby Magnetically Activated Cell Sorting (MACS), first by depleting CD45⁺cells from the lower band of a 30%/70% Percoll gradient followed bypositive selection for EpCAM⁺ cells (FIG. 6B). Purity of the resultingcell suspensions was >95%. c, Relative to CD45−/EpCAM− cells,CD45−/EpCAM+ cells expressed high levels of EpCAM, CK19, Trop2, andCD133 and low levels of AFP, LGR5, CD90, and Vimentin (FIG. 6C). Albuminwas expressed at comparable levels in CD45⁻ /EpCAM⁻ and CD45⁻/EpCAM⁺fractions.

Example 3 Notch Signaling in Liver Progenitors In Vitro

To elucidate the role of Notch signaling in liver regeneration, an invitro culture system was developed. Cell cultures were established byculturing primary oval cells on Sw-3T3 fibroblasts (ATCC), arrested withMitomycin C (Sigma), in High Glucose Dulbecco's Modified Eagle Medium(DMEM; Invitrogen) supplemented with 15% Fetal Calf Serum (Sigma),non-essential amino acids (Invitrogen), Glutamax (Invitrogen), and ITS(Invitrogen).

The anti-activated Notch2 antibody (clone 40-2-7) was generated againstthe peptide VIMAKRKRKHGSLW, corresponding to amino acids 1697-1710 ofthe human Notch2 protein sequence (SEQ ID NO:73), coupled to KLH (YenZymCustom Antibodies, LLC). Splenocytes from a rabbit producing an antibodywith the appropriate specificity were used to generate hybridomas(Epitomics, Inc.). Clone 40-2-7 was identified by screening theresulting rabbit monoclonal antibodies by immunoblotting andimmunohistochemistry. This antibody recognizes both human (FIG. 7B, leftpanel) and mouse (FIG. 7B, right panel) active Notch2 at endogenouslevels.

Progenitors cultured in the culture system maintained growth (FIG. 2A)and formed colonies consisting of small, tightly packed cells with ahigh nuclear-cytoplasmic ratio and a distinct raised edge (FIG. 2A, leftpanel). The cells within these colonies were uniformly EpCAM positive(FIG. 2A, right panel). Progenitor cultures also maintained thecharacteristic progenitor expression signature in vitro (FIG. 2C).Activated Notch2 was detected by Western blot analysis using a rabbitmonoclonal antibody raised against the S3 cleaved form of the humanNotch2 protein (FIG. 7B). Activated Notch2 signal was increased uponligand stimulation (Jag) or EDTA stimulation (EDTA), and the activatedform was greatly enriched in the nuclear fraction (FIG. 7B; lanes “N”).

Treatment of the primary cultures with the γ-secretase and Notch pathwayinhibitor N—[N-(3,5-difluorophenacetyl)-1-alanyl]-S-phenylglycinet-butyl ester (DAPT) unexpectedly enhanced colony formationapproximately ten-fold, from approximately 1 colony formed per 100,000plated CD45 negative, non-parenchymal cells to approximately 1 colonyper 10,000 cells plated (FIG. 2B) suggesting that inhibition of Notchpathway activity promoted either liver progenitor cell maintenance orproliferation. Treatment with DAPT resulted in a small increase inprogenitor cell proliferation (FIG. 2D).

To determine if Notch signaling inhibition also suppresses thedifferentiation of progenitor cells, thereby allowing for long-termmaintenance, the biliary and hepatic differentiation potential of thesecells in vitro was analyzed (FIG. 7). Cultured oval cells weremaintained on a mitomycin-C treated feeder layer of Swiss-3T3fibroblasts by culture in the presence of 15% Fetal Bovine Serum (FBS)and the γ-secretase inhibitor DAPT.

Differentiation along the hepatocyte lineage was induced by plating ovalcells without feeder cells onto tissue-culture treated plastic coatedwith diluted Rat Tail Collagen (BD) in the presence of 10 ng/mlOncostatin M (R&D) and 25 ng/mL HGF (Lonza). For some experiments, DAPTor vehicle (DMSO) and/or the anti-Notch2 NRR antibody, Antibody D-3(also referred to herein as anti-N2, anti-Notch2, or anti-NRR2) or anisotype control antibody were added to the medium at the time of cellplating and replenished every three days. Bile duct differentiation wasinduced by suspending oval cells in a 1:1 mixture of MATRIGEL and ovalcell growth medium supplemented with 7.5% FBS and plating onto plastictissue culture dishes. Following solidification, the MATRIGEL cultureswere overlain with growth medium supplemented with 15% FBS. In someexperiments, DAPT or vehicle and/or anti-Notch2 antibody or isotypecontrol were added to the MATRIGEL as well as the overlying medium,which was replenished every three days.

Progenitor cells that were cultured on a collagen substrate in thepresence of Hepatocyte Growth Factor and Oncostatin M displayed achanged cellular morphology consistent with hepatocyte differentiation,including larger cell size, lower nuclear-cytoplasmic ratio, and twonuclei (FIG. 2E-F). Hepatocyte-associated transcripts Albumin anda-Fetoprotein (AFP; FIG. 2G) were also increased in these cells. Notch2signaling was active in cultured oval cells, as transcriptionally activeNotch2 intracellular domain (ICD) could be detected using an antibodyspecific to the γ-secretase cleaved form of this receptor (FIG. 2H). Theappearance of the cleaved form was dependent on γ-secretase activity, asit was absent from DAPT treated cells (FIG. 2H). Specific binding of ananti-Notch2 inhibitory antibody also blocked formation of this activeform of Notch2 (FIG. 2H). As expected, treatment with the Notch2inhibitory antibody leads to a decrease in Notch target gene Hes1(p=6E-05) (FIG. 2I). Surprisingly, progenitor cells that were culturedon a collagen substrate in the presence of Hepatocyte Growth Factor andOncostatin M in the presence of an anti-Notch2 inhibitory antibody (Wuet al., Nature 464(7291):1052 (2010)) (FIG. 2H-I) resulted in a morepronounced hepatocyte-like morphology (FIG. 2 K) and increased albuminexpression level (FIG. 2L), compared to cells cultured with the isotypecontrol (FIG. 2J, L).

In contrast, differentiation of cultured oval cells along the biliarylineage, assessed in three dimensional culture, was decreased byinhibition of Notch2. Cells cultured in the presence of an anti-Notch2inhibitory antibody displayed a less differentiated morphology (FIG. 2N)and a 50% decreased expression of the biliary marker Keratin 19 (CK19)(p=2E-05) (FIG. 2O) compared to cells grown in the absence of theinhibitory antibody (FIG. 2M, O). Together, these results are consistentwith the notion that Notch2 inhibition biases differentiation away fromthe biliary lineage and toward hepatocyte formation.

Example 4 Inhibition of Notch2 Signaling In Vivo

To determine the effect of Notch2 inhibition in liver damage, a rodentmodel of liver damage was employed. Mice were partially hepatectomizedby removal of left lateral and median lobes according to Yokoyama et al.(Yokoyama et al., Cancer Research 13(1):80-85 (1953)), which results incompensatory proliferation of hepatocytes and recovery of liver masswithin 7-10 days (Higgins and Anderson, Arch. Pathol., 12:186 (1931);Yokoyama et al., Cancer Res. 13(1):80 (1953)). Mice were injectedintraperitoneally with an anti-Notch2 NRR antagonist antibody (Wu etal., Nature 464(7291):1052 (2010)) or an anti-Ragweed isotype controlantibody at a dose of 5 mg/kg twice per week, including twice prior tohepatectomy. Two hours prior to liver harvest, mice were injectedintraperitoneally with Bromodeoxyuridine at 50 mg/kg.Immunohistochemistry was performed on 5 μm liver sections usingantibodies for BrdU (DAKO), pan-Cytokeratin (WSS; DAKO), and Hes1 (MBLInternational). Antibody binding was detected using standardstreptavidin-HRP/DAB for BrdU and pan-Cytokeratin and tyramide signalamplification (TSA)/DAB for Hes1.

Treatment with anti-Notch2 antibody resulted in effective Notch2inhibition as determined by significant splenic Marginal Zone B-cell(MZB) depletion (p<0.0001, FIG. 8A-C) (Wu et al.). MZB representapproximately 5% of the splenic B lymphocytes in normal C57Bl/6 mice.Treatment with Notch2 antagonist (5 mg/kg, 2×/week) resulted in avirtual disappearance of MZB (FIG. 8A-C). Notch2 inhibition wasmaintained through the course of partial hepatectomy experiments asindicated by persistent depression in MZB population (FIG. 8C).Inhibition of Notch2 did not significantly alter the rate of liver massrecovery immediately following partial hepatectomy (FIG. 8D), which inearly stages is due largely to hepatocyte hypertrophy and division ofpre-existing polyploid hepatocytes (St. Aubin and Bucher, The AnatomicalRecord 112(4):797 (1952); Higgins and Ingle, The Anatomical Record,73(1):95 (1939)). Notch2 inhibition caused a small decrease in overallBrdU incorporation at 40 hours (FIG. 8E; (p=0.027)) and a much largerand more significant decrease in BrdU incorporation in intrahepatic bileducts (FIG. 8F), suggesting that anti-Notch2 treatment affected liverprogenitor cells within the bile ducts. Consistent with thisobservation, expression of the Notch target gene Hes1 was detectedprimarily in intra-hepatic bile duct cells (FIG. 3C-1) rather than inhepatocytes. Moreover, the percent Hes1-positive intrahepatic bile ductcells was significantly reduced in anti-Notch2 treated mice 40 hoursafter surgery, compared to control antibody treated mice, and in manyportal areas Hes1 staining was not observed (FIG. 3C-2). Despite thereduction in Hes1-positive cells, bile duct morphology was unaffected(FIG. 3, compare panels A-1-D-1 on left (isotype) to panels A-2-D-2 onright (anti-Notch2)) and no significant elevation of markers of biliarydysfunction was observed, even after one month of ongoing Notch2inhibition (FIG. 11F-H).

Example 5 Effects of Notch2 Inhibition on Liver Regeneration In Vivo

To determine whether Notch2 inhibition affects the recovery ofhepatocyte function following partial hepatectomy, serum hepatobiliaryfunction markers were assessed following ⅔ partial hepatectomy in micetreated with anti-Notch2 or control antibody. Unexpectedly, recovery inliver function began earlier in anti-Notch2-treated animals compared tocontrols. Serum albumin levels started increasing by 40 hours postsurgery (FIG. 9A) and reached significantly (p<0.05) higher levels inanti-Notch2 antibody-treated mice at both the 40 hour and 72 hour timepoints (FIG. 3E; FIG. 9A), compared to serum levels in control animalsthat started to increase by 72 hours after surgery (2.8 versus 2.4 g/dL,p<0.02). This improvement in recovery of pre-operative serum albuminlevels was accompanied by reduced expression of Hes1 (p<0.02; FIG. 3F).These results suggest that Notch2 inhibition results in enhancedrecovery of hepatocyte function. Markers of hepatocyte damage were notsignificantly different between treatment and control groups (FIG.5B-F). However, a consistent and significant increase in the ratio ofalbumin to K19 transcripts, referred to herein as DifferentiationQuotient, was observed (FIG. 10E). The average Differentiation Quotientof anti-Notch2 antibody-treated livers, normalized to pre-surgicallevels, recovered more quickly, regaining 75% of pre-surgical values 3days after surgery and 100% of pre-surgical values between 3 and 6 daysafter surgery (FIG. 10E). In contrast, Differentiation Quotient valuesin isotype control antibody-treated animals did not recover topre-surgical levels until 14 days after surgery (FIG. 10E). Theseresults suggest that inhibition of Notch2 biases the differentiation ofbipotent liver progenitor cells away from the biliary (K19-positive)towards the hepatic (albumin-positive) lineage. Differences betweenanti-Notch2 and isotype control antibody-treated livers in apparent denovo hepatocyte formation could be detected as early as 24 hours afterpartial hepatectomy. Transcript levels of in the form of the immaturehepatocyte marker alpha-fetoprotein (AFP) were significantly elevated inanti-Notch2 antibody-treated mice (FIG. 10B). Also, morphological andfunctional differences between anti-Notch2 and control antibody-treatedlivers persisted for up to 2 weeks after surgery as the anti-Notch2antibody-treated livers appeared more robust, with a noticeably moreuniform parenchymal architecture (FIG. 3A-2) compared to control-treatedlivers (FIG. 3A-1).

The effects of Notch2 inhibition on liver regeneration were also studiedin a 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) model of chronicliver disease. This DDC model is known in the art as a model of chronicliver disease. The mechanism of liver damage in response to DDC isabnormal heme metabolism with accumulation of protoporphyrin which istoxic to the hepatocytes. Thus, the DDC model can also serve as modelfor hereditary or acquired defects in the heme metabolic pathway.

C57BL/6N female mice 8-12 weeks of age (Charles River) were fed acholine deficient diet (20% Lard; Teklad TD.04523) supplemented with0.15% (w/v) Ethionine (supplier) in the drinking water to induce ovalcells (Akhurst et al., Hepatology 34(3):519 (2001)). The prolongedhepatotoxic influence of a DDC diet led to a proliferation ofcytokeratin (CK)19-positive progenitor cells (FIG. 3D-1), referred to asoval cell response, reminiscent of the ductular reaction common in humanhepatobiliary disease (Farber, Cancer Research, 16(2):142 (1956)). Afterfour weeks of DDC, the oval cell reaction had increased such thatCK19-positive oval cells occupied an average of about 15% (10-20%) ofthe total hepatic cross sectional area (FIG. 3D-1; FIG. 3G), while serummarkers of hepatobiliary injury were greatly elevated (p<0.0001, FIG.3H; FIG. 11A-H). However, treatment with the anti-Notch2 inhibitoryantibody significantly impeded the oval cell reaction (FIG. 3D-2),reducing the average cross-sectional area of CK19-positive tissue fromapproximately 15% to only 5% of total liver cross sectional area(p<0.0001, FIG. 3G). Despite this striking reduction in oval cellproliferation, hepatic architecture was not adversely affected byanti-Notch2 antibody treatment and was grossly indistinguishable fromcontrol-treated tissue (FIG. 3D-2). The decrease in CK19-positive ovalcells associated with Notch 2 inhibition was accompanied by improvedhepatobiliary function, with significantly decreased total and directserum bilirubin levels, a marker of cholestasis and other forms ofhepatobiliary damage (p=0.0003, FIG. 3H). Also, the DifferentiationQuotient was significantly elevated in livers from mice treated with theanti-Notch2 antibody (p<0.0001, FIG. 3J) suggesting improved hepatocytefunction Inhibition of Notch2 signaling by treatment with an anti-Notch2antibody was confirmed by a greater than 70% reduction in Hes1expression in the biliary and progenitor cells in anti-Notch2antibody-treated livers (p<0.0001, FIG. 3I), reflecting the central roleof Notch2 signaling in governing oval cell fate choice.

Taken together, these results show that treatment with anti-Notch2 NRRantibody facilitates the recovery of liver function after two differenttypes of liver damage, one by partial hepatectomy and one by chemicaldamage (choline-limiting diet). Mechanistically, anti-Notch2 NRRantibody facilitates liver recovery by favoring hepatocytedifferentiation and by preventing aberrant (or pathologic) bile ductproliferation. Accordingly, treatment with anti-Notch2 NRR antibodycould, e.g., prevent progression of chronic liver disease, such as liverfibrosis.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, the descriptions and examples should not be construed aslimiting the scope of the invention. The disclosures of all patent andscientific literatures cited herein are expressly incorporated in theirentirety by reference.

1. A method of treating a liver condition characterized by liver damage,the method comprising administering to a patient having such conditionan effective amount of a Notch2-specific antagonist.
 2. The method ofclaim 1, wherein the liver condition is chronic liver disease.
 3. Themethod of claim 2, wherein the chronic liver disease is selected fromthe group consisting of liver fibrosis, cirrhosis, viral hepatitis,autoimmune liver diseases, genetic liver diseases, alcoholic hepatitisand nonalcoholic fatty liver disease.
 4. The method of claim 3, whereinthe autoimmune liver disease is selected from the group consisting ofautoimmune hepatitis, primary biliary cirrhosis, and primary sclerosingcholangitis.
 5. The method of claim 1, wherein the liver condition is anacute liver condition.
 6. The method of claim 5, wherein the acute livercondition is acetaminophen toxicity.
 7. The method of claim 1, whereinthe Notch2-specific antagonist is selected from the group consisting ofa soluble Notch receptor, soluble Notch ligand variant, aptamer,oligopeptide, anti-Notch2 antagonist antibody, and anti-Notch2 ligandantagonist antibody.
 8. The method of claim 7, wherein the anti-Notch2antagonist antibody is an anti-Notch2 negative regulatory region (NRR)antibody.
 9. The method of claim 8, wherein the anti-Notch2 NRR antibodybinds to the Lin 12/Notch Repeat-A and heterodimerization domain-Cdomains of Notch2 NRR.
 10. The method of claim 8, wherein theanti-Notch2 NRR antibody comprises: (a) a heavy chain hypervariableregion (HVR-H) 1 comprising an amino acid sequence of SEQ ID NO:3; (b)an HVR-H2 comprising the amino acid sequence of SEQ ID NO:4; (c) anHVR-H3 comprising the amino acid sequence of SEQ ID NO:5; (d) a lightchain hypervariable region (HVR-L) 1 comprising an amino acid sequenceof SEQ ID NO:10; (e) an HVR-L2 comprising an amino acid sequence of SEQID NO:14; and (f) an HVR-L3 comprising an amino acid sequence of SEQ IDNO:19.
 11. The method of claim 8, wherein the anti-Notch2 NRR antibodycomprises an HVR-H1 comprising an amino acid sequence selected from SEQID NOs:1-2; an HVR-H2 comprising the amino acid sequence of SEQ ID NO:4;an HVR-H3 comprising the amino acid sequence of SEQ ID NO:5; an HVR-L1comprising an amino acid sequence selected from SEQ ID NOs:6-9; anHVR-L2 comprising an amino acid sequence selected from SEQ ID NOs:11-13;and an HVR-L3 comprising an amino acid sequence selected from SEQ IDNOs:15-18.
 12. The method of claim 7, wherein the anti-Notch2 antagonistantibody is an anti-Notch2 antibody that binds to one or more EGF-likerepeats of Notch2.
 13. The method of claim 1, wherein theNotch2-specific antagonist is administered by intramuscular,intraperitoneal, intracerobrospinal, subcutaneous, intra-articular,intrasynovial, intrathecal, oral, topical, or inhalation route.
 14. Amethod of inducing hepatic differentiation, the method comprising thestep of contacting an oval cell with an effective amount of aNotch2-specific antagonist, thereby inducing hepatic differentiation ofthe oval cell.
 15. The method of claim 14, wherein the oval cell iscontacted with the Notch2-specific antagonist in vitro.
 16. The methodof claim 14, wherein the oval cell is contacted with the Notch2-specificantagonist in vivo.
 17. A method of reducing aberrant bile ductproliferation in a patient in need thereof, the method comprising thestep of administering to the patient an effective amount of aNotch2-specific antagonist, thereby reducing aberrant bile ductproliferation.
 18. The method of claim 3, wherein the genetic liverdiseases is selected from the group consisting of alpha-1 antitrypsindeficiency, Crigler-Najjar syndrome, familial amyloidosis, Gilbert'ssyndrome, Dubin-Johnson syndrome, hereditary hemchromatosis, primaryoxalosis, and Wilson's disease.
 19. The method of claim 5, wherein theacute liver condition is selected from the group consisting of acuteliver failure, acute liver injury, and acute liver toxicity.
 20. Themethod of claim 1, 8 or 9, wherein administering to the patient theNotch2-specific antagonist results in accelerated differentiation ofhepatocyte progenitor cells into hepatocytes compared to hepatocyteprogenitor cell differentiation in the absence of the Notch2-specificantagonist.
 21. The method of claim 1, 8 or 9, wherein administering tothe patient the Notch2-specific antagonist results in decreased aberrantbile duct proliferation compared to aberrant bile duct proliferationwithout administering the Notch2-specific antagonist.
 22. The method ofclaim 1, 8 or 9 wherein administering to the patient the Notch2-specificantagonist results in improved liver histological appearance compared toliver histological appearance without administering the Notch2-specificantagonist.
 23. The method of claim 22, wherein the improved liverhistologic appearance is selected from the group consisting of increasedcell size, decreased nuclear-to-cytoplasmic ratio and increased numberof liver cells having two nuclei.
 24. The method of claim 1, 8 or 9,wherein administering to the patient the Notch2-specific antagonistresults in decreased expression of Keratin-19 in liver cells relative toexpression of Keratin-19 in cultured adult oval cells.
 25. The method ofclaim 1, 8 or 9, wherein administering to the patient theNotch2-specific antagonist results in increased expression of albumin inliver cells relative to expression of albumin in cultured adult ovalcells.
 26. The method of claim 1, 8 or 9, wherein administering to thepatient the Notch2-specific antagonist results in increased expressionof α-fetoprotein in liver cells relative to expression of α-fetoproteinin cultured adult oval cells.
 27. The method of claim 1, 8 or 9, whereinadministering to the patient the Notch2-specific antagonist results in areduced number of Hes1-positive intrahepatic bile duct cells.
 28. Themethod of claim 1, 8 or 9, wherein administering to the patient theNotch2-specific antagonist results in reduced serum bile acids, serumbilirubin, or serum alkaline phosphatase.
 29. The method of claim 8,wherein the anti-Notch2 NRR antibody further comprises at least oneframework selected from a human variable heavy acceptor 2 framework anda human variable light kappa subgroup I consensus framework.
 30. Themethod of claim 8, wherein the anti-Notch2 NRR antibody comprises: (a)an HVR-H1 comprising the amino acid sequence of SEQ ID NO:1; (b) anHVR-H2 comprising the amino acid sequence of SEQ ID NO:4; (c) an HVR-H3comprising the amino acid sequence of SEQ ID NO:5; (d) an HVR-L1comprising the amino acid sequence of SEQ ID NO:6; (e) an HVR-L2comprising the amino acid sequence of SEQ ID NO:11; and (f) an HVR-L3comprising the amino acid sequence of SEQ ID NO:15.
 31. The method ofclaim 8, wherein the anti-Notch2 NRR antibody comprises: (a) an HVR-H1comprising the amino acid sequence of SEQ ID NO:2; (b) an HVR-H2comprising the amino acid sequence of SEQ ID NO:4; (c) an HVR-H3comprising the amino acid sequence of SEQ ID NO:5; (d) an HVR-L1comprising the amino acid sequence of SEQ ID NO:7; (e) an HVR-L2comprising the amino acid sequence of SEQ ID NO:11; and (f) an HVR-L3comprising the amino acid sequence of SEQ ID NO:16.
 32. The method ofclaim 8, wherein the anti-Notch2 NRR antibody comprises: (a) an HVR-H1comprising the amino acid sequence of SEQ ID NO:2; (b) an HVR-H2comprising the amino acid sequence of SEQ ID NO:4; (c) an HVR-H3comprising the amino acid sequence of SEQ ID NO:5; (d) an HVR-L1comprising the amino acid sequence of SEQ ID NO:8; (e) an HVR-L2comprising the amino acid sequence of SEQ ID NO:12; and (f) an HVR-L3comprising the amino acid sequence of SEQ ID NO:17.
 33. The method ofclaim 8, wherein the anti-Notch2 NRR antibody comprises: (a) an HVR-H1comprising the amino acid sequence of SEQ ID NO:2; (b) an HVR-H2comprising the amino acid sequence of SEQ ID NO:4; (c) an HVR-H3comprising the amino acid sequence of SEQ ID NO:5; (d) an HVR-L1comprising the amino acid sequence of SEQ ID NO:9; (e) an HVR-L2comprising the amino acid sequence of SEQ ID NO:13; and (f) an HVR-L3comprising the amino acid sequence of SEQ ID NO:18.
 34. The method ofclaim 8, wherein the anti-Notch2 NRR antibody comprises a heavy chainvariable domain having at least 90% sequence identity to an amino acidsequence selected from SEQ ID NO:20-21 and a light chain variable domainhaving at least 90% sequence identity to an amino acid sequence selectedfrom SEQ ID NO:22-25.
 35. The method of claim 34, wherein the heavychain variable domain comprises the amino acid sequence of SEQ ID NO:21,and the light chain variable domain comprises an amino acid sequenceselected from SEQ ID NOs:23-25.
 36. The method of claim 35, wherein theheavy chain variable domain comprises the amino acid sequence of SEQ IDNO:21, and the light chain variable domain comprises an amino acidsequence of SEQ ID NOs:25.
 37. The method of claim 8, wherein theanti-Notch2 NRR antibody competes for binding with an antibodycomprising a heavy chain variable domain comprising the amino acidsequence of SEQ ID NO:21, and a light chain variable domain comprisingan amino acid sequence selected from SEQ ID NOs:23-25.
 38. The method ofclaim 14, wherein the Notch2-specific antagonist is an anti-Notch2antagonist antibody.
 39. The method of claim 38, wherein the anti-Notch2antagonist antibody is an anti-Notch2 NRR antibody.
 40. The method ofclaim 39, wherein the anti-Notch2 NRR antibody binds to the Lin 12/NotchRepeat-A and heterodimerization domain-C domains of Notch2 NRR.
 41. Themethod of claim 17, wherein the Notch2-specific antagonist is selectedfrom the group consisting of a soluble Notch receptor, soluble Notchligand variant, aptamer, oligopeptide, anti-Notch2 antagonist antibody,and anti-Notch2 ligand antagonist antibody.
 42. The method of claim 41,wherein the anti-Notch2 antagonist antibody is an anti-Notch2 NRRantibody.
 43. The method of claim 42, wherein the anti-Notch2 NRRantibody binds to the Lin 12/Notch Repeat-A and heterodimerizationdomain-C domains of Notch2 NRR.
 44. A method of reducing liverprogenitor cell proliferation in a patient in need thereof comprisingthe step of administering to the patient having such condition aneffective amount of a Notch2-specific antagonist.
 45. The method ofclaim 44, wherein the Notch2-specific antagonist is selected from thegroup consisting of a soluble Notch receptor, soluble Notch ligandvariant, aptamer, oligopeptide, anti-Notch2 antagonist antibody, andanti-Notch2 ligand antagonist antibody.
 46. The method of claim 45,wherein the Notch2-specific antagonist is an anti-Notch2 antagonistantibody.
 47. The method of claim 46, wherein the anti-Notch2 antagonistantibody is an anti-Notch2 NRR antibody.
 48. The method of claim 47,wherein the anti-Notch2 NRR antibody binds to the Lin 12/Notch Repeat-Aand heterodimerization domain-C domains of Notch2 NRR.